Performance of anaerobic digesters

ABSTRACT

Methods of improving anaerobic digestion by enhancing the microorganism population through the creation of a nutrient environment more favorable for microbial growth and through the addition of microbes and, more particularly, by the addition of chelating agents, chelating agents and nutrients, and methanogens. Methods for the improved production of said methanogens by the provision of controlled amount of methanol and H 2  and CO 2 . Methods for the improved preservation of said methanogen by ultra-freezing and freeze-drying.

FIELD OF THE INVENTION

The invention relates generally to anaerobic digestion processes for thetreatment and stabilization of organic wastes and more specifically tomethods of improving anaerobic digestion by enhancing the microorganismpopulation through the creation of a nutrient environment more favorablefor microbial growth and through the addition of microbes.

BACKGROUND AND INTRODUCTION TO THE INVENTION

Anaerobic digestion is a biological process which can be used to convertinexpensive, readily available and inexhaustable resources, notablysewage sludge, industrial and municipal waste and biomass material tovaluable product gases, principally methane. Anaerobic digestion hasbeen used commercially for the treatment and stabilization of organicwastes for a century. Almost every municipality disposes of most of itssewage sludge by anaerobic digestion. In addition, many industries treatsolid and liquid waste streams in anaerobic treatment facilities. Theprocess has advantages, not only in its suitability for treating wasteshaving a high water content or difficulties in incineration, but alsofacilitates the recovery of methane as a source of clean energy. A greatdeal of effort has gone into designing anaerobic digestion systems whichoperate reliably, efficiently and quickly. Among the designs developedfor anaerobic digestion systems are the completely mixed reactor, theanaerobic contact process, the upflow and downflow packed bed reactors,the fluidized bed reactor, the upflow anaerobic sludge blanket, thebaffled reactor, and various two stage processes. (The apparatusemployed in carrying out the anaerobic digestion processes are referredto as reactors or digesters.)

All of these processes are dependent upon the same anaerobicmicroorganisms to perform the conversion of the organic matter tomethane. While Applicant does not wish to be bound by any particulartheory or hypothesis with regard to this discussion or any otherthroughout this application, anaerobic digestion of organic matter tomethane and carbon dioxide is presently thought to occur in threestages, each of which involves a different type of bacteria.

The first stage of anaerobic digestion involves the fermentativebacteria. Fermentative bacteria hydrolyze the polymers of the primarysubstrates, such as polysaccharides, proteins and lipids, and fermentthem to produce fatty acids (such as propionate and butyrate), organicacids, alcohols, ammonia, sulfide, CO₂ and H₂.

The second stage of anaerobic digestion is the degradation of the fattyacids, some organic acids and alcohols produced in the first stage.These compounds are degraded by a second group of bacteria called theobligate H₂ -producing (i.e., proton-reducing) acetogenic bacteria. Onlya few of these microorganisms have been isolated and studied. Theproducts of the second stage degradation include acetic acid, H₂ andCO₂.

The third and final stage of anaerobic digestion involves themethanogenic bacteria. Some methanogenic bacteria cleave acetic acid toproduct CO₂ and CH₄. This reaction is very important becauseapproximately 70% of the methane produced during anaerobic digestion isderived from the methyl group of acetic acid. Other methangenic bacteriautilize the H₂ produced by other microorganisms to reduce CO₂ and CH₄.

Depending on the content of the waste material fed into an anaerobicdigester, different stages may be the rate-limiting step in thedigestion process. In the digestion of soluble compounds, therate-limiting step has been identified as the third stage ormethanogenesis, and more specifically, the aceticlastic (uses aceticacid) methanogenic step. For the digestion of insoluble wastes, thehydrolysis of these materials, or the first stage of anaerobicdigestion, may be the rate-limiting step.

Hydrogen, which is involved in many principal biological reactions, isrecognized as being the controlling influence on the overall scheme ofwaste utilization. Hydrogen exerts control at certain points ofanaerobic digestion.

Hydrogen plays an important role in regulating the proportions of thevarious products produced by the fermentative bacteria. Duringfermentation, H₂ is produced from electrons generated in the oxidationof reduced pyridine nucleotides:

    NADH+H.sup.+ ⃡H.sub.2 +NAD.sup.+ ΔG°'=18.0kj/reaction

H₂ formation is favored only when the partial pressure of H₂ is verylow, as it is when H₂ is effectively metabolized by methanogens duringthe third stage of anaerobic digestion. At low partial pressures of H₂,the flow of electrons (NADH) generated during glycolysis is toward thereduction of protons, resulting in H₂ formation. The H₂ allows pyruvateto be degraded to acetate, CO₂ and H₂. When the partial pressure of H₂is increased, the flow of electrons from NADH shifts from H₂ productionto the formation of reduced electron sink fermentation products, such aspropionate and longer-chained fatty acids, lactate or ethanol frompyruvate. Therefore, in ecosystems in which methanogens are effectivelyutilizing H₂, fermentative bacteria produce more acetate, CO₂ and H₂,but produce little or no ethanol or lactate and produce considerablyless propionate and butyrate.

Hydrogen also exerts control during the process in which acetogenicbacteria act on fatty acids, organic acids and alcohols to produceacetic acid and hydrogen. The equilibrium reactions for the degradationof butyrate and propionate under standard conditions are:

    CH.sub.3 CH.sub.2 CH.sub.2 COO.sup.- +2H.sub.2 O⃡2CH.sub.3 COO.sup.- +H.sup.+ +2H.sub.2

    ΔG°'++48.1 kJ/reaction

    CH.sub.3 H.sub.2 COO.sup.- +3H.sub.2 O⃡CH.sub.3 COO.sup.- +H.sup.+ +2H.sub.2

    ΔG°'=+76.1 kJ/reaction

As indicated by large positive values for ΔG°', these reactions do notfavor degradation. However, when the partial pressure of H₂ ismaintained at a very low level in the digester, H₂ production from thesecompounds is thermodynamically favorable. For example, when the partialpressure of H₂ is decreased below 0.15 atm, the degradation of ethanolbecomes energetically favorable. However, the degradation of butyrate orpropionate is not energetically favorable until the partial pressure islowered to about 2×10⁻³ atm or 9×10⁻³ atm, respectively. Thus, a slightincrease in the partial pressure of H₂ will halt the degradation ofthese compounds, with propionate degradation being the first reaction tobe inhibited. As expected, studies have shown that during digesterfailure, propionate is the first acid that accumulates.

Thus, as discussed above, the partial pressure of H₂ not only regulatesthe proportion of end products of the fermentative bacteria in stage onebut also the degradation of these fermentation products by the H₂-producing acetogenic bacteria in stage two.

Methanogenic bacteria, in addition to producing gaseous fuel (methane)in stage three, can be used to maintain the H₂ concentration in thereactor at a very low level and thus allow the digestion of organicmatter to proceed efficiently. Therefore, to obtain maximum performancefrom an anaerobic digester, the concentration of methanogens within thedigester should be maximized. Several methods have been used to obtainhigh concentrations of bacterial biomass (including high concentrationsof methanogens) in anaerobic digesters. These methods include: (1) therecycle of solids in the anaerobic contact process; (2) attachment ofbiomass to supports in fixed films and fluidized or expanded beds; and(3) formation of granular biomass in a sludge blanket. While thesemethods have been successful in achieving high bacterial concentrations,they do not increase th population of methanogens in proportion to theother bacteria. Hence, the H₂ partial pressures are not regulated inthese systems and rates are not maximized.

The reaction kinetics of anaerobice digestion are quite slow. Reactiontimes of up to 30 days may be required to efficiently stabilize andreduce sludge volumes and/or reduce the Biological Oxygen Demand (BOD)to acceptable levels. In addition, the start-up of anaerobic digestersfor sludge treatment is a slow and uncertain process. It may takeseveral months after inoculation before a digester is operating at itsdesired capacity. In some cases, for example, in a very large fixed filmreactor, it may take up to one year for the anaerobic biomass to developsufficiently to attain maximum digestion efficiency.

Once a digester has achieved maximum efficiency, if the digester isshocked, for example, by shortening the retention time, overloading thesystem or adding a toxic substance, recovery can also be very slow. Asanaerobic digestion is presently practiced, it may take several monthsto recover from the resulting lowered conversion efficiency or completeinhibition of conversion and regain the conversion efficiency whichexisted before the upset.

SUMMARY OF THE INVENTION

The present invention is directed to a method of improving anaerobicdigestion by enhancing the microorganism population through the creationof a nutrient environment more favorable for microbial growth ofmethanogenic bacteria. Preferred techniques for increasing theconcentration of methanogens in anaerobic digester sludge include theaddition of chelating agents to the sludge, in order to increase thesolubility of inorganic nutrients, and the addition of chelating agentsalong with selected inorganic nutrients. The present invention is alsodirected toward methods of methanogen addition which not onlysubstantially enhance the rate of anaerobic digestion, but also aid insmooth and rapid digester start-up, as well as recovery after upset.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows methane production of methanogenic bacteria as a functionof time for control and nitrilotriacetate (NTA) addition.

FIG. 2 shows methane production of methanogenic bacteria as a functionof time for control and methanogen (Methanosarcina barkeri) amendedculture.

FIG. 3 is an illustration of the laboratory reactor used for continuousculture experiments.

FIG. 4 shows cumulative gas production of methanogenic bacteria as afunction of time for control and methanogen (Methanosarcina barkeri)amended reactors.

FIG. 5 shows cumulative gas production during recovery of upsetdigesters. Cumulative gas production (L[stp]) is shown as a function oftime for control and Methanosarcina barkeri (MB) amended reactors.

FIG. 6 shows gas production improvement with ethylenediaminetetra-acetic acid (EDTA) amendment. The percent improvement in gasproduction is shown as a function of dosage of EDTA.

FIG. 7 shows gas production improvement with citric acid (CA) amendment.The percent improvement in gas production is shown as a function ofdosage of CA.

FIG. 8 shows the average gas production improvement for chelating agentamendment, i.e., the average improvement for amendment with EDTA or CA.The percent improvement in gas production is shown as a function ofdosage of chelating agent.

FIG. 9 shows average gas production and improvement as a function ofretention time. The percent improvement in gas production (left-handy-axis) and average gas production (right-hand y-axis) are shown as afunction of retention time in days.

FIG. 10 shows projected annual savings for EDTA amendment. Annualsavings (in $1,000/year) is shown as a function of dosage of EDTA.

FIG. 11 shows annual savings for CA amendment. Annual savings (in$1,000/year) is shown as a function of dosage of CA.

FIG. 12 shows gas production of the control reactor in the 1 g/Lmethanogen test.

FIG. 13 shows gas production of 1 g/L methanogen amended reactor.

FIG. 14 shows gas production of control reactor in 2 g/L methanogentest.

FIG. 15 shows gas production of 2 g/L methanogen amended rector.

FIG. 16 shows gas production of control reactor in upset and 3 g/L test.

FIG. 17 shows gas production of upset and 3 g/L amended reactor.

FIG. 18 shows the reaction rate coefficient for digester 1.

FIG. 19 shows the reaction rate coefficient for digester 2.

FIG. 20 shows percent conversion as a function of inverse retention timefor digeser 1.

FIG. 21 shows percent conversion as a function of inverse retention timefor digester 2.

DETAILED DESCRIPTION OF THE INVENTION Addition of Chelating Agents Alone

Methanogenic bacteria require iron, cobalt, nickel and molybdenum forgrowth and methanogenesis. Although these trace metals may be found inmost digesters in sufficient quantity to allow growth of methanogens,they may be precipitated and/or complexed with other elements, thusmaking them unavailable for uptake by methanogenic bacteria.

The present invention provides methods for increasing the availabilityof these trace metals to methanogenic bacteria by solubilizing the tracemetals. Preferably, the trace metals are solubilized by the addition ofchelating agents. A partial list of chelating agents and their stabilityconstants is given in Table 1. For the chelating agents listed in Table1, the affinity is generally greatest for Fe³⁺ and least for calcium andmagnesium. The degree of binding of iron depends on whether it ispresent in ferric (Fe³⁺) or ferrous (Fe²⁺) form. In addition, agentswhich form lipophilic complexes, for example, EDDHA and8-hydroxyquinoline, may permeate the plasma membrane of an organism andassist in the uptake of metal ions.

                                      TABLE 1                                     __________________________________________________________________________    Stability constants of some chelating agents of importance in culture         media                                                                                   Log stability constant                                              Chelating agent                                                                         Fe.sup.3+                                                                        Cu.sup.2+                                                                         Zn.sup.2+                                                                         Co.sup.2+                                                                         Fe.sup.2+                                                                         Mn.sup.2+                                                                         Ca.sup.2+                                                                        Mg.sup.2+                                 __________________________________________________________________________    EDDHA.sup.(2)                                                                           33.9                                                                             15  9.3 --  14.3                                                                              --  7.2                                                                              2.9                                       CDTA.sup.(1)                                                                            27.5                                                                             21.3                                                                              18.5                                                                              18.9                                                                              16.3                                                                              14.7                                                                              12.5                                                                             10.3                                      EDTA.sup.(1)                                                                            25.1                                                                             18.3                                                                              16.3                                                                              16.2                                                                              14.3                                                                              13.6                                                                              10.7                                                                             8.7                                       NTA.sup.(1)                                                                             15.9                                                                             12.8                                                                              10.5                                                                              10.6                                                                              8.8 7.4 6.4                                                                              7.0                                       Histidine.sup.(1)                                                                       -- 18.3*                                                                             12.9*                                                                             13.9*                                                                             9.3*                                                                              7.7*                                                                              -- --                                        8 Hydroxyquinoline                                                                      26.3*                                                                            25.4*                                                                             17.1*                                                                             19.5*                                                                             15.0*                                                                             13.5*                                                                             13.2*                                                                            12.0*                                     1:10 Phenanthroline                                                                     14.1*                                                                            18.0                                                                              17.0*                                                                             --  21.0*                                                                             7.4*                                                                              -- --                                        2:2' Dipyridyl                                                                          -- 17.9                                                                              13.5                                                                              --  17.6                                                                              6.3 -- --                                        SSA       14.1                                                                             9.4 --  6.5 --  5.3 -- --                                        Glycine.sup.(1)                                                                         -- 15.2*                                                                             9.5*                                                                              8.9*                                                                              7.8*                                                                              4.7*                                                                              -- --                                        Citric acid.sup.(1)                                                                     11.4                                                                             5.9 5.0 5.0 4.4 3.7 3.6                                                                              3.3                                       Polyphosphate                                                                           6.5*                                                                             5.5*                                                                              6.0*                                                                              3.0 3.0 5.5*                                                                              3.0                                                                              3.2                                       __________________________________________________________________________     EDDHA = ethylenedinitriloN,N'-bis(2'hydroxyphenyl)-N,N'-diacetic acid         CDTA = 1:2 diaminocyclohexaneN,N-tetra-acetic acid                            EDTA = ethylenediamine tetraacetic acid                                       NTA = nitrilotriacetic acid                                                   polyphosphate = [P.sub.n O.sub.3n+1 ].sup.(n+2)- where n ≈ 5          SSA = 5sulphosalicylic acid                                                   The stability constant is K = [ML]/[M][L] where [ML], [M] and [L] are the     concentrations of complex, free metal ion and free ligand respectively        *Stability constant is β.sub.2 = [ML.sub.2 ]/[M][L].sup.2, which is      obtained from the equilibrium M + 2L ⃡ ML.sub.2                    Stability constant is β.sub.3 = [ML.sub.3 ]/[M][L].sup.3, which is      obtained from the equilibrium M + 3L ⃡ ML.sub.3 -            

Applicant has discovered that the addition of certain chelating agentsto an anaerobic digestion system results in increased production ofmethane, as well as increased destruction of solids. The results ofApplicant's experiments examining effect of addition of chelating agentson the growth of methanogenic bacteria are described in Example 1. Fourchelating agents were utilized: 1:2 diamino cyclohexane-N,N-tetra aceticacid (CDTA), nitrilotriacetate (NTA), ethylenediamine tetra-acetic acid(EDTA), and citric acid (CA).

Applicant found that in all cases in which chelating agents were added,methane production increased. As shown in Tables 4, 6, 8 and 10, theincrease varied from 5 to 20 percent, depending on the chelating agentadded, with NTA addition resulting in a 20 percent improvement. Theresults of the NTA experiments, which are shown graphically as afunction of time in FIG. 1, demonstrate that methane production isconsistently higher than the control and the improvement over controlincreases with time.

Applicant further discovered that the rate of methane production isenhanced with the addition of chelating agents. For example, in the EDTAexperiment, at 9 days the EDTA culture had produced 6.1×10⁻³ gmol/L ofmethane. By contrast, it took 15 days for the control culture to producethis quantity of methane. This represents a rate increase of 40 percent.In the NTA experiment, the methane production rate improved from 1.23ml/day to 1.52 ml/day with NTA addition, which represents a 23 percentincrease after 15 days.

Applicant also found that the addition of chelating agents resulted in asubstantial increase in the destruction of solids. See Tables 3, 5, 7and 9.

In summary, the results of the experiments described in Example 1 showthat methane production and solids destruction are stimulated by theaddition of chelating agents. Moreover, the production tends to increasewith increasing concentrations of NTA, EDTA, and CA. The rate ofproduction is also stimulated, showing a definitive improvement afterone day and significantly faster rates during the 15 days.

The stimulation effect is much more pronounced for NTA than for theother chelating agents. For example, methane production is increased byabout 20 percent after 15 days. This magnitude of increased methaneproduction shows that the chelating compound is not simply serving as anadditional carbon source for methane production. The theoreticalconversion of 1 uM of NTA into methane is about 4.5 micromoles ofmethane. However, the 1 uM experiment shows an increase in methaneproduction of about 500 micromoles over the control. Therefore, the100-fold increase in methane production could not be due to metabolismof NTA to methane. Applicant believes the stimulation results from thechelating agent's ability to increase the solubility of essentialinorganic nutrients which promote the growth and stability of themethanogenic population.

Thus, Applicant has demonstrated that, by the addition of relativelysmall amounts of chelating agents, methane production from anaerobicdigesters can be increased or, alternatively, smaller digesters can beused to achieve the same methane production. Also, faster digesterstart-up, greater digester stability and more rapid digester recoveryfrom upsets should result from the addition of chelating agents.

Addition of Nutrients Alone

Iron, sulfide, selenium and nickel are required by methanogenic bacteriafor growth and methanogenesis, i.e., the production of methane. Iron andsulfide are particularly important to the rapid development of thebacteria. For example, when Methanobacterium bryantii strain M.o.H.,which utilizes H₂ and CO₂ to produce methane, is grown with only 0.007g/l FeSO₄.7H₂ O, its generation time is in excess of 30 hours. However,when the FeSO₄.7H₂ O is increased to 0.01 g/l, as was used in generatingthe data in Table 2, the generation time is decreased to 6 hours.

Table 2 also shows that the growth rates for a wide number ofmethanogens are quite slow and limit the retention times at which mostanaerobic digesters can operate. Consequently, anaerobic digestersoperate at lenthy retention times to maintain bacterial growth and anadequate methanogen population. Therefore, substantial opportunity forimprovement is possible.

The data in Table 2 were obtained under a H₂ :CO₂ atmosphere with 2.5percent sodium acetate as a cell carbon source, 0.125 percent cysteine(added as a reducing agent) and trace vitamins being the only organicconstituents of the medium. When organics such as trypticase and yeastextract are added, the generation time is somewhat shortened.

To further illustrate the importance of iron in the growth ofmethanogens, Table 2 shows growth data for Methanogenium olentangyi,Methanococcus deltas and Methanospirillum hungatei. These organismswould not grow at all unless the iron concentration were at least 0.01g/l.

Applicant theorized that the potential existed to reduce the retentiontime and to enhance the growth and numbers of methanogens in anaerobicdigesters, given the proper nutrient environment within the reactor.

                  TABLE 2                                                         ______________________________________                                        Growth Characteristics of Fourteen Methanogens                                                         Max O.D.                                             Organism          g(h).sup.a                                                                           580 nm    .sup.Y methane.sup.b                       ______________________________________                                        Methanobrevibacter smithii PS                                                                   10.0   1.1 ± .06                                                                            1.08 ± .06                              Methanobacterium thermoauto-                                                                    1.6    1.1 ± 14                                                                             0.73 ± .09                              trophicum H                                                                   Methanobacterium thermoauto-                                                                    3.0    1.0 ± .06                                                                            0.78 ± .11                              trophicum RC                                                                  Methanobacterium formicicum                                                                     6.0    1.2 ± .06                                                                            1.07 ± 04                               MF1                                                                           Methanobacterium formicicum RC                                                                  3.8    1.3 ± 12                                                                             1.15 ± .05                              Methanosarcina barkeri MS                                                                       7.0    N.D..sup.c                                                                              2.14 ± .24                              Methanosarcina barkeri 227                                                                      7.6    N.D.      2.27 ± .14                              Methanocuccus vannielii                                                                         16.0   0.7 ± .03                                                                            1.60 ± .11                              Methanospirillum hungatei JF                                                                    13.0   0.8 ± .05                                                                            0.84 ± .12                              Methanocuccus maripaludis                                                                       3.0    0.9 ± .02                                                                            1.47 ± .04                              Methanobacterium bryantii M.o.H.                                                                6.0    1.0 ± .1                                                                             1.30 ± .09                              Methanobacterium olentangyi                                                                     11.0   0.9 ± .01                                                                            1.06 ± .04                               Methanococcus deltae                                                                           2.0    0.9 ± .07                                                                            1.41 ± .19                              Cuyahoga River coccus                                                                           11.6   0.9 ± .03                                                                            1.01 ± .03                              ______________________________________                                         .sup.a g(h) indicates generation time in hours.                               .sup.b .sup.Y methane equals the gram dry weight of cells per mole of         methane produced.                                                             .sup.c N.D. not determined.                                              

The availability of sulfide is also of great importance to the growth ofmethanogens. Sulfide is a required nutrient for all methanogens and iscritical to the aceticlastic methanogen Methanosarcina barkeri. It isnecessary to balance the addition of trace metals and sulfide which, ifadded in excess, may precipitate as salts and cause sulfide to beunavailable to methanogens for uptake.

The importance of nickel and selenium to stimulate the growth ofmethanogens has also been recognized.

Applicant conducted experiments to determine the effect of addition oftrace quantities of inorganic nutrients as described in Example 2. Theinorganic nutrients utilized were nickel in the form of NiCl₂, iron inthe form of FeSO₄ and FeCl₂, and selenium as SeO₂. Tables 11-18 presentthe data for the cultures with added nutrients. These data are presentedon the same basis as for the chelating studies.

Applicant demonstrated that, in all cases, the volatile solidsdestruction was reduced and methane production was either unaffected orimpaired by the addition of nutrients. In no case was the methaneproduction enhanced. Significant suppression did not result for nutrientconcentrations of 100 uM or less. Methane production was repressed 5-8percent at the 1000 uM concentration.

The data are quite conclusive. Addition of excess nutrients in the formof NiCl₂, FeSO₄, FeCl₂, or SeO₂ did not improve methane production andactually repressed methane production at high nutrient concentrations. Apossible explanation is that these metals may be complexing with sulfidein the sludge to form insoluble sulfides. Evidence of precipitatedsulfides (dark color) was apparent in those experiments with addednutrients. Sulfide is an extremely important nutrient for the organismsinvolved in an anaerobic digestion culture. The effect of precipitatinginsoluble sulfides is to reduce the availability of free sulfide to themicroorganisms, such that sulfide becomes limiting. When sulfide islimiting, the culture becomes inefficient in producing methane, evenwhen other nutrients are present in excess.

Addition of Chelating Agents and Inorganic Nutrients

Applicant-conducted experiments to determine the effect of addinginorganic nutrients with chelating compounds to an anaerobic digestionculture. In these experiments, described in Example 3, the nutrientFeSO₄ was added alone or along with either chelator CA or CDTA.

Tables 19 and 20 show the results obtained for the addition of 1000 uMFeSO₄ alone and with 20 uM CA or CDTA. As before, the addition ofnutrient only resulted in suppression of the methane formation andreduction of volatile solids destruction. However, unexpectedly, theaddition of a small quantity of chelating compound with FeSO₄ resultedin increased methane production over the control. It appears that thechelated iron did not precipitate other essential nutrients and thusallowed them, as well as the iron, to remain available for microorganismgrowth. The greater effectiveness of CDTA over CA can probably beattributed to its higher log stability coefficient for Fe²⁺ (16.3 vs.4.4).

Addition of Methanogens

As discussed above, the methanogens can function to keep the hydrogenconcentration within a reactor very low, allowing the fermentativebacteria to produce a large percentage of acetate and H₂ :CO₂, and a lowpercentage of propionate, lactate, methanol and butyrate. A low hydrogenconcentration also enables the obligate proton-reducing acetogens toconvert butyrate and propionate to acetate, H₂, and CO₂.

Applicant has demonstrated that the addition of methanogens to a reactorsubstantially enhances the rate of anaerobic digestion. Applicant hasalso demonstrated that the addition of small numbers of methanogens to astressed reactor balances the fermentation and allows rapid recovery.Additionally, Applicant has shown that added methanogens shortendigester start-up time and serve to establish methanogens in highnumbers to handle the products of the fermentative and acetogenicbacteria. The methods used to evaluate the effect of addition ofmethanogens on digester performance are described in Examples 4-10.

Initially, batch experiments were carried out as described in Example 4.In these experiments, pure cultures of methanogens grown in a mineralsalts medium without rich organics were used for injection into bottlesof sludge. The following methanogens were used:

Methanosarcina barkeri (MB)

Methanobrevibacter smithii (MS)

Methanobacterium formicium (MF)

The results of Applicant's studies on the addition of these threemethanogens to batch cultures in various combinations are given inTables 21 and 22. In all cases, the addition of methanogens resulted inan increase in solids destruction and an increase in methane production.This supports the theory that methanogens reduce the H₂ concentrationwith a resultant improvement in efficiency.

The most significant improvement was for Methanosarcina barkeri (MB)which yielded an overall 11 percent increase in methane generation after18 days. The MB-amended sludge showed a 28 percent increase in methaneproduction over the control in only one day. These results are showngraphically in FIG. 2. For the MB culture, the rate of methaneproduction increased dramatically for the first five days, after whichthe rate of improvement became less pronounced. The amended culturesustained the higher level of production and continued to improve overthe duration of the experiment. The MB is the only methanogen testedcapable of aceticlastic methanogenesis (as well as H₂ :CO₂ reductivemethanogenesis). However, Applicant's hypothesize that the major reasonfor its superior performance may be due to a higher cell concentration,rather than its ability to perform aceticlastic methanogenesis, becauseit grew to higher cell densities than did the other methanogens.

By these experiments, Applicant confirmed that methanogenesis is therate limiting step in sludge digestion. Applicant showed that reactorsamended with pure cultures of methanogens could produce higherquantities of methane and reduce sludge disposal volumesproportionately. In addition, Applicant showed that the addition ofmethanogens could facilitate start-up of digesters and recovery fromupsets.

The performance of an anaerobic digestion culture can be best evaluatedunder continuous operation, rather than by batch experiments. Undercontinuous operation, the microorganism population reaches a stableconcentration. At this point, the effect of amendments (e.g., additionof methanogens) can be more accurately measured. Also, continuousoperation more nearly mimics industrial applications.

Semi-continuous reactors, as described in Example 5 and illustrated inFIG. 3, were used to evaluate the enhancement of a digester start-upwith the addition of methanogens. The results of the experiments aregiven in Table 23. Weekly gas productions at Standard Temperature andPressure (STP) are given for the four-week operating period. Methaneconcentrations ranged from 55-65 percent of total gas produced.Applicant discovered that the amended culture consistently produced from10-30 percent more gas than the control. These results are showngraphically in FIG. 4, where the cumulative gas production is plotted asa function of time. The MB-amended culture produced an average of 24percent more methane than the control. This improvement would beexpected to be even greater if the retention time were longer.

As discussed above, once an anaerobic digester has achieved optimalefficiency, if it is shocked, such as by overload of the system,addition of a toxic substance, or shortening of retention time, recoverycan be very slow. Applicant performed experiments to determine whetheradded methanogens could stimulate recovery of a shocked digester. Theexperiments are described in Example 6.

An upset digester was simulated by pH adjustment to cause inhibition ofdigestion. The methanogens were then added to the reactor to determinethe stimulation from recovery. A control consisted of an uninoculatedsludge reactor which was not upset. The same six parameters as werefollowed in the start-up experiments described in Example 5 werefollowed here as well.

Applicant has shown that the addition of methanogens substantiallyenhances the recovery of a shocked anaerobic digester. Such upsets arenot uncommon in practice, thus, this type of amendment has significantpotential for commercial application. Perhaps more significant is theshowing in FIG. 5 that the rate of gas production was increasing fasterafter two weeks in the digester with amendment than in the controlreactor. This result again indicates that a significant improvement inoperation and gas production might be achieved on a continuous basis.

In summary, Applicant's experiments demonstrated that the addition ofpure methanogens to anaerobic digestion cultures enhanced the methaneproduction by 11 percent in a batch culture, 24 percent in a continuousreactor after start-up and nearly double during recovery from an upset.Because methane production is considered to be the rate limiting step insludge digestion, some enhancement in the kinetics was to be expected.However, the magnitude and duration of the enhancement demonstrated inthe continuous experiments was not expected. These data indicate that asubstantial permanent enhancement can be achieved with the addition ofsmall quantities of methanogens.

Chelation Optimizing Experiments

To confirm that the results of the batch chelation experiments would beapplicable to commercial anaerobic digesters, which are operatedsemi-continuously, Applicant conducted additional continuousexperiments.

These experiments under continuous culture conditions also served todetermine the optimal levels and schedule of chelating agents to beadded to anaerobic digesters to maximize methane production and sludgedestruction. The experiments are described in Example 7 and the resultsof Applicant's studies to determine optimal dosages of chelating agentsare summarized in Table 25 and FIGS. 6-9.

These data show that substantial improvements are possible at the usualoperating conditions and indicate that low dosages of chelating agentsmay be optimal. As shown in FIG. 6 and 7, respectively, the improvementin gas production vs. dosage of EDTA and CA is more sensitive to theretention time than to the chelating agent concentration. The longer theretention time, the greater the improvement in gas production. Theresults indicate that at low retention time (high flow rates), there areadequate dissolved nutrients to support microbial gorwth. However, atlonger retention times, the available nutrients are insufficient tosupport maximum growth without the addition of chelating agents.

As shown in FIG. 8, the optimal improvement in methane (gas) productionis achieved with a 10 uM dosage of chelating agent. Although someenhancement is obtained by using dosages above 10 uM, the additionalimprovement is only about 3 percent per 100 uM.

As noted above, the improvement in gas production is sensitive to theretention time. In fact, as can be seen from FIG. 9, the improvement isproportional to the retention time. FIG. 9 show data for retention timesof 10, 15 and 25 days. However, because most commercial reactors operateat retention times of 25 days or longer, the possibility for evengreater enhancement of gas production exists.

The selection of the optimal chelator and dosage can be based simply onthe annual savings from addition of the amendment. One such analysis isdescribed in Example 8. FIGS. 10 and 11 show the net benefit of the EDTAand CA amendment at different retention times. Since the unit cost ofEDTA is less than half that of CA, the influence of the dosage to thenet benefit was quite different with these two chelating agents. The lowcost of EDTA makes the amendment under higher dosages advantageous.However, low dosage amendment is more attractive in the case of CA. Theoptimal condition is 500 uM dosage of EDTA at a 15 day retention time,which results in an annual savings of $15,000. However, the betterchoice in practice may be a 10 uM dosage at the same retention time,which results in $12,800/yr benefit with annual cost of EDTA of $88,instead of $4,108 with the 500 uM dosage. The best condition for the CAamendment is with 10 uM dosage at a 25-day retention time, which resultsin an $8,800/yr savings.

Extended Continuous Methanogen Experiments

Extended continuous methanogen experiments were conducted by Applicantto determine the level of enhancement of methanogen addition tooperating digesters and to determine the effect of methanogen additionon recovery after upset.

As shown in Example 9, the improvement of 1 g/L Methanosarcina barkeriamendment at a 25-day retention time averaged 35% higher gas productionover the control reactor for more than five months operation. Thisimprovement appears to be permanent. The use of an additional 1 g/Lamendment increased gas production to 59% over the control for anotherfour-month operation period. Again, the improvement seems permanent at a25-day retention time..

Applicant believes that these significant improvements are the result ofthe ability of Methanosarcina barkeri to consume both acetate and H₂.The H₂ partial pressure regulates the proportions of the variousproducts from the fermentative bacteria in the first stage and controlsthe degradation of the fatty acids to acetate in the second stage. Thus,low H₂ pressures maintained by Methanosarcina barkeri improve the ratesof these stages of the process.

Therefore, the addition of methanogens does improve the overall processrate, and suprisingly, the improvement is relatively large andpermanent.

It has long been thought that in sludge digestion, the rate limitingstep was methanogensis. The increased methanogen population functions tospeed up the rate limiting step, as well as the other steps. However,Applicant found the increase to be nearly 60 percent at long retentiontimes and lasted for nearly one year. Even greater improvement mightresult with an increased dosage.

The permanent effect might be explained by considering that a normalculture can only move to a higher methanogen population by anover-production of acetate as substrate for methanogen growth. An excessAmount of acetate would lower the pH and adversely affect methanogenperformance and growth. Thus, a normal culture might never evolve to ahigher methanogen population, given the many other variables that arechanging. However, addition of methanogens would allow an increase inthe acetate production to sustain the higher cell concentration.

As stated earlier, the significant improvement of the amended reactor isattributed to the ability of Methanosarcina barkeri to consume bothacetate and H₂. Through the use of labeled acetic acid, it has beenfound that 73 percent of the methane produced in a standard anaerobicdigester comes from acetate. Thus, the utilization of acetate byMethanosarcina barkeri plays an important role in the improvement.Furthermore, the bacteria are also responsible for maintaining the H₂Concentration in the reactor at a very low level in order for thefermentation to proceed efficiently. Hydrogen is involved in manyprincipal biological reactions and is recognized as being thecontrolling influence on the overall scheme of anaerobic digestion.Hydrogen exerts an important influence in controlling the proportion ofproducts from the first stage of anaerobic digestion, as well as thedegradation of these products in the second stage. Therefore,Methanosarcina barkeri, which uses both H₂ and acetate, enhances thefermentation by maintaining lower H₂ levels, as well as producing moregas from acetic acid.

To determine the ability of a reactor amended with methanogens torecover from an upset, an additional experiment was performed asdescribed in Example 10. An imposed upset with sulfuric acid for one dayinhibited the gas production in both control and amended reactors forthree week. After the third week, 1 g/L more of methanogens wasinoculated. The gas production from both reactors increased to aboutdouble the amount before the upset. This condition remained for twomonths before the control reactor returned to its normal performance.After such time, the improvement of the amended reactor was about 60%over the control.

Field Trails

To further demonstrate the commercial feasibility of the present methodsfor improving performance of anaerobic digesters, Applicant conductedfield trials. The field experiments were conducted at the Springdale,Arkansas Waste Treatment Plant, which has two primary anaerobicdigesters. The volumes of Digesters 1 and 2 are 683,300 and 767,162gallons, respectively. There are no gas meters installed or any means ofdetermining the gas production rates. Since gas measurement is notpossible, improvement in performance was measured as increased solidsdestruction. The experiments, which are described in Example 11, werebased on the amendment of 10 uM CA to one digester, while keepinganother digester as the control.

The amendment of Digester 1 of the Springdale Waste Treatment plant with10 uM CA resulted in improvement of the gas production by about 5%.However, the average temperature was reduced about 4.5° F. during thistest. Therefore, under normal conditions, the actual improvement mightbe much better.

A 10 uM C-A amendment of Digester 2 resulted in a 10% improvement.

Considerable variability in retention time, temperature, and sludgequality was encountered in these field experiments. Nevertheless, 5-10%improvements in reaction rates were observed with only 10 uM amendment.

Methanogen Production

Although the concentrations are small (1 g/L), large quantities of puremethanogenic bacteria will be required for amending commercialdigesters. Experiments have been conducted to determine appropriatemethods for growth and preservation of methanogens.

Methanosarcina barkeri was chosen for the studies of methanogenproduction, which are described in Example 12, because of its ability toutilize acetate and H₂ and because of the good results obtained inamending reactors with this organism. Methanosarcina barkeri grows on H₂-CO₂, CO, methanol, methaylamines and acetate. Growth of Methanosarcinabarkeri on a mixture of methanol and acetate has been determined to bediauxic, methane being produced from methanol and acetate in sequence;in the presence of H₂ --CO₂ and acetate, the former substrate has beenshown to be preferentially used. The simultaneous use of H₂ --CO₂ andmethanol have been observed during growth of Methanosarcina barkeri withmethanol contributing to greater methane production. It has been shownthat methanol is used for methanogenesis at a rate five times that ofother substrates and that methanol is readily converted to methane inthe presence of H₂ --CO₂, without a lag phase. The methane production isabout the sum of the production from methanol and H₂ --CO₂ . Therefore,the supply of methanol under an atmosphere of H₂ --CO₂ should providefor rapid production of cells. However, if acetate is not present whenthe Methanosarcina barkeri culture depletes H₂, these cultures may losethe ability to use acetate as the sole substrate. Therefore, acetate isalso provided in the medium. The medium used is listed in Table 34.

Although methanol has been reported as the preferred carbon and energysource for the growth of Methanosarcina barkeri, the gas composition wasalso found to significantly influence the growth. Two batch experimentswere conducted to identify the appropriate methanol and gascompositions. Rapid growth of Methanosarcina barkeri was obtained byusing methanol as the major substrate in fed-batch operation. However,80% H₂ and 20% CO₂ gas is also required to support this rapid growthrate. The growth is inhibited when the methanol concentration is higherthan 1.2%.

The supply of H₂ --CO₂ gas was found to influence the growth rate. Ifthe gas is supplied too often, a faster initial growth rate results, butcannot be sustained. If the gas is supplied too slowly, growth isimpaired and methanol inhibition results. Thus, the supply of the gasshould be precisely controlled.

The best production condition in batch culture was found to be provisionof 1% methanol every two days, with the addition of 20 psig 80% H₂ and20% CO₂ every two days. The maximum wet cell concentrations were 19.25,35.0, 38.71, 40.41, and 43.75 g/L after 3, 6, 9, 12, and 15 daysproduction, respectively. Further improvement of these results should bepossible by close control of the methanol concentration. Theconcentration of the initial inocula can also be increased for morerapid growth. Production should be easily improved to 40 g/L within oneweek.

As noted above, both the methanol addition and the interval of H₂ andCO₂ re-pressurization were important factors in the growth ofMethanosarcina barkeri. However, the addition of the methanogen mediumor concentrated medium had no significant effect. Generally, methanoladdition at the rates of 0.6% per day or 1.0% every two days gave thebest results. The best interval for H₂ and CO₂ re-pressurization wasevery two days. While re-pressurization every day helped the cell growthin the initial, stage, high growth rates were not sustained. Since H₂and CO₂ are also carbon and energy sources, there is competition betweenthese gases and methanol. When the gas is over-supplied, as in the caseof re-pressurization every day, the methanol consumption is reduced andmethanol concentrations accumulate. When inhibition levels are reached,the growth stops. On the other hand, if the supply of gases is notenough as with re-pressurization every three days, insufficient growthoccurred, and methanol accumulated and stopped growth. With H₂ and CO₂addition every two days, and 0.6% per day methanol addition, a cellconcentration of 30 g/L was reached after six days of fermentation. Themaximum cell concentration achieved was 38 g/L after 15 days by theaddition of 1% methanol every two days. The limitation of cellconcentrations to these levels could be caused by methanol accumulation,lack of some critical nutrient, or cell age or density limitations.

It has been reported that an excess of H₂ inhibits methanogenesis frommethanol. In the current experiment, the 80% H₂ and 20% CO₂ gascomposition used was controlled by the flow from two gas cylinders. Thegas controls may not have been accurate and an excess of H₂ waspossible. The use of a custom graded 80% H₂ and 20% CO₂ gas cyliner gavebetter results than the flow controlled mixing. Further studies examinedthe effect of adding 1.0% methanol every two days with gas additionevery two days, adding 0.4% and no methanol every day withre-pressurization every day, and addition of 0.3 ml of 10% yeast extractat day 9. These results are given in Table 40. The best result foundpreviously was a cell concentration of 38.5% g/L at day 15. With thepremixed gas cylinder, the growth was improved to 43.75 g/L at day 15.The cell concentration was 38.7 g/L at day 9, before addition of theyeast extract.

For the 0.4% and no methanol addition, the cell growth was faster thanwith flow controlled mixing of the gas. However, lower cellconcentrations resulted from these conditions. The cell growth wassignificantly stimulated by yeast extract in the 0.4% methanolexperiment. However, no significant stimulation resulted in the 1%methanol case. The conclusion of these experiments is that rapid cellgrowth can be achieved in semi-batch culture by adding 1% methanol and20 psig H₂ --CO₂ every two days. It is also concluded that methanogenproduction can be improved by the provision of 0 to 1.2% by volumemethanol in the liquid phase and 80% H₂ and 20% CO₂ by volume in the gasphase. These conditions provide the basis for potential large scaleproduction.

Methanogen Preservation

If methanogen amendment of anaerobic digesters is to be practicedcommercially, methods for storage and shipment of methanogens must bedeveloped. The viability of methanogens stored with differentpreservation methods was investigated.

Applicant's experiments, which are described in Example 13, demonstratedthat both freeze-drying to at least minus 40° C. and ultra-freezing toat least minus 70° C. can be used for the preservation of methanogens.After three months storage, the methanogens preserved by freeze-dryingwith 24% sucrose or 20% skim milk (as cryoprotection) and byultra-freezing with 5% DMSO or 10% glycerol (as cryoprotection) or withno cryoprotection all reached the viability of unpreserved methanogens.However, based on the time required to reach unpreserved viability, thepreferable preservation method is ultra-freezing with 5% DMSO.

EXAMPLES

The following examples are set forth to assist in-understanding theinvention and should not, of course, be construed as specificallylimiting the invention described and claimed herein. Such variations ofthe invention as would be within the purview of those skilled in theart, including the substituion of equivalents now known or laterdeveloped, are to be considered to fall within the scope of theinvention as hereinafter claimed.

EXAMPLE 1 ADDITION OF CHELATING AGENTS--BATCH EXPERIMENTS

Anaerobic sludge was obtained from a local municipal waste watertreatment plant. A 29 ml aliquot was anaerobically dispensed into 125 mlcrimp seal serum bottles. Flanged thick butyl rubber stoppers held inplace by aluminum crimp seals were used to insure anaerobic conditionsand prevent gas leaks. The chelators to be tested were made up inanaerobic solutions and injected by syringe into the bottles. Thefollowing chelating agents were tested in concentrations of 1 uM, 10 uM,100 uM and 1000 uM:

1. Nitrilotriacetate (NTA)

2. Ethylenediamine tetraacetic acid (EDTA)

3. Citric acid (CA)

4. 1:2 diamino cyclohexane -N, -N-tetraacetic acid (CDTA)

These agents have log stability constants for Fe³⁺ ranging from 27.5 forCDTA to 11.4 for citric acid. Effectiveness may vary between agents.Even though the chelating agent binds a trace metal strongly, it maystill be available to the methanogen by virtue of being in solution.

The following parameters were followed as a function of time for 15days:

1) gas volume production

2) methane production

3) carbon dioxide production

4) PH--initial and final

5) Volatile solids--initial and final

6) Volatile fatty acids--initial and final

Control samples with no additions, and samples with varyingconcentrations of chelating agents were run in triplicate. The resultsfor the CDTA, NTA, EDTA, and CA experiments are given in Tables 3-10.Tables 3, 5, 7, and 9 show the initial and final (after 15 days)concentrations of total solids, volatile solids, and ash, as well as thetotal volume of gas produced for each of the concentration levelsutilized. The characteristics of the beginning sludge inoculum are givenin the first row for each experiment. Volatile fatty acids were alsomeasured but were present in insignificant concentrations in allexperiments. Tables 4, 6, 8, and 10 give the methane production from thetime of inoculation for the various chelating agents and concentrations.These methane quantities appear small but are normal considering the lowsubstrate concentration. These values are presented as the methaneconcentrations in the gas space of the serum bottle, and an increase inconcentration, of course, denotes an increase in methane production. Theconcentration of CO₂ was not measured daily, but when measured showedthat the gas produced was 55-70 percent methane.

                  TABLE 3                                                         ______________________________________                                        Results of CDTA Amended Cultures                                              (after 15 days - all data are average of 3 replicates)                               Dry          Volatile Gas Production (ml)                              Amend-   Solids  Ash    Solids Mea-  Calculated                               ment   pH    g/L     g/L  g/L    sured V.S. Methane                           ______________________________________                                        Begin- 7.2   9.127   2.932                                                                              6.195  0     0    0                                 ning                                                                          Control                                                                              7.1   7.276   2.780                                                                              4.496  35.5  28.3 24.8                                1 uM 7.8   7.208   2.748                                                                              4.460  36.2  43.6 26.3                               10 uM 7.7   7.132   2.688                                                                              4.444  40.0  44.0 27.5                               100 uM                                                                              7.7   7.132   2.708                                                                              4.424  31.7  44.6 27.6                              1000 uM                                                                              7.7   7.260   2.636                                                                              4.624  32.7  38.9 26.5                              ______________________________________                                    

                  TABLE 4                                                         ______________________________________                                        Methane Production from CDTA Amended Cultures                                            Methane Production (10.sup.-3 mole/L)                                         Day                                                                Amendment    4     6           9   15                                         ______________________________________                                        Control      2.2   3.9         5.1 5.0                                          1 uM       2.3   4.2         5.1 5.0                                         10 uM       2.7   4.3         5.1 5.2                                         100 uM      2.3   4.1         5.1 5.2                                        1000 uM      2.5   4.3         5.2 5.2                                        ______________________________________                                    

                  TABLE 5                                                         ______________________________________                                        Results of NTA Amended Cultures                                               (after 15 days - all data are average of 3 replicates)                                             Vol-                                                             Dry          atile   Gas Production (ml)                              Amend-    Solids  Ash    Solids                                                                              Mea-  Calculated                               ment   pH     g/L     g/L  g/L   sured V.S. Methane                           ______________________________________                                        Begin- 7.60   7.312   2.499                                                                              4.813 0     0    0                                 ning                                                                          Control                                                                              7.68   6.607   2.447                                                                              4.160 22.7  14.0 28.6                                1 uM 7.61   6.304   2.300                                                                              4.004 24.7  18.48                                                                              31.9                               10 uM 7.67   6.318   2.300                                                                              4.018 24.7  18.09                                                                              32.2                               100 uM                                                                              7.67   6.397   2.343                                                                              4.054 25.6  17.06                                                                              32.8                              1000 uM                                                                              7.68   6.291   2.421                                                                              3.870 27.5  22.30                                                                              36.6                              ______________________________________                                    

                  TABLE 6                                                         ______________________________________                                        Methane Production from NTA Amended Cultures                                             Methane Production (10.sup.-3 mole/L)                                         Day                                                                Amendment    4     6           9   15                                         ______________________________________                                        Control      2.1   4.2         4.6 6.9                                          1 uM       2.2   4.3         4.7 7.4                                         10 uM       2.3   4.4         4.8 7.5                                         100 uM      2.3   4.4         4.9 7.7                                        1000 uM      2.4   4.5         5.0 8.2                                        ______________________________________                                    

                  TABLE 7                                                         ______________________________________                                        Results of EDTA Amended Cultures                                              (after 15 days - all data are average of 3 replicates)                                         Dry           Volatile                                                                              Gas                                                     Solids   Ash  Solids  Production                             Amendment pH     g/L      g/L  g/L     ml                                     ______________________________________                                        Control   7.45   7.284    2.565                                                                              4.719   76.7                                     1 uM    7.40   7.342    2.577                                                                              4.765   70.0                                    10 uM    7.40   7.265    2.539                                                                              4.726   70.0                                    100 uM   7.37   7.363    2.588                                                                              4.775   69.3                                   1000 uM   7.35   7.575    2.860                                                                              4.715   77.7                                   ______________________________________                                    

                  TABLE 8                                                         ______________________________________                                        Methane Production from EDTA Amended Cultures                                             Methane Production (10.sup.-3 g mole/L)                                       Day                                                               Amendment     4     6          9    15                                        ______________________________________                                        Control       5.4   6.1        14.4 16.1                                        1 uM        5.5   6.2        15.2 16.3                                       10 uM        5.6   6.5        15.7 16.5                                       100 uM       5.8   6.8        15.9 16.7                                      1000 uM       5.6   10.6       16.3 16.7                                      ______________________________________                                    

                  TABLE 9                                                         ______________________________________                                        Results of CA Amended Cultures                                                (after 15 days - all data are average of 3 replicates)                                             Vol-                                                             Dry          atile   Gas Production (ml)                              Amend-    Solids  Ash    Solids                                                                              Mea-  Calculated                               ment   pH     g/L     g/L  g/L   sured V.S. Methane                           ______________________________________                                        Begin- 7.54   10.905  3.189                                                                              7.716 0     0    0                                 ning                                                                          Control                                                                              7.54   8.797   2.851                                                                              5.946 57.0  43.1 47.2                                1 uM 7.49   8.832   2.864                                                                              5.968 61.0  42.5 48.1                               10 uM 7.53   8.777   2.852                                                                              5.925 59.7  43.7 48.5                               100 uM                                                                              7.51   8.801   2.868                                                                              5.933 61.0  43.5 50.0                              1000 uM                                                                              7.53   8.976   3.052                                                                              5.924 62.3  43.7 50.1                              ______________________________________                                    

                  TABLE 10                                                        ______________________________________                                        Methane Production from CA Amended Cultures                                                Methane Production (10.sup.-3 g mole/L)                                       Day                                                              Amendment      4     6          9    15                                       ______________________________________                                        Control        4.6   7.7        9.1  9.0                                        1 uM         48    7.9        9.4  9.2                                       10 uM         4.8   8.1        9.6  9.2                                       100 uM        5.4   8.3        9.8  9.5                                      1000 uM        5.6   8.6        10.1 9.5                                      ______________________________________                                    

As noted, the solids concentrations differ between experiments and,consequently, the gas production and methane production differ. Eachexperiment was started with fresh inoculum at different times,therefore, the concentrations were dependent upon the samples obtainedfrom the treatment plant. Consequently, comparison between tables forthe various amendments should be based upon the improvement over thecontrol in each experiment.

The accuracy and precision of the data in Tables 3-10 can be determinedby material balance on the samples. For example, Table 3 shows that theinitial volatile solids concentration of the sludge was 6.195 g/L. After15 days, the volatile solids concentration had been reduced to anaverage of 4.496 g/L in the control samples. Therefore, 0.0298 g ofvolatile solids (after correction for dilution) was converted to gas.Using a conversion factor of 0.95 L gas/g solids destroyed (based on anaverage sludge composition of 50 percent carbon), the gas producedshould equal 28.3 ml. The actual gas production measured was 35.5 ml, sothat reasonable agreement is obtained. It should be noted that gasvolumes are measured in a syringe by equilibrating the pressure in theserum bottle. This method of measurement is standard practice for smallgas volumes, but allows some error, depending upon the friction in thesyringe barrel.

Another check of the consistency of the data can be made from themethane production. The methane concentration in the serum bottle gasspace is measured by gas/solid chromatography. These measurements aremade periodically during the 15 day period and show the rate andquantity of methane produced. Established chromatography methods areused and these measurements should be accurate and reliable. Again,using data from the control samples of CDTA in Table 4, the methaneconcentration is 0.005 gmol/L. Multiplying by the volume of 130.5 ml(gas space plus gas produced) gives a methane production of 0.000653gmol or a methane volume of 14.9 ml. Since the methane is about 60percent of the gas production, the total gas produced is 24.8 ml, whichagrees reasonably well with the 28.3 ml computed from volatile solidsdestruction.

Values of the calculated gas production from volatile solids and methaneare given in the tables. It is concluded that the data are consistentbetween solids destruction and methane and total gas production.Although slight variations may be apparent, the data of the tables aresufficiently accurate to demonstrate trends and draw conclusions.

EXAMPLE 2 Addition of Inorganic Nutrients Alone

These experiments were conducted as in Example 1, except that nutrientswere added in place of the chelating agents. The experiments werecarried out in batch culture in 125 ml serum bottles with an inoculum of29 ml of sludge from a waste water treatment plant and one ml ofnutrient medium. The characteristics of the sludge vary depending uponthe day of collection and are given in the first row of the followingtables. The following nutrients were added in the followingconcentration ranges:

    ______________________________________                                        Nutrient(s)   Concentration range                                             ______________________________________                                        FeCl.sub.2    1-5000 uM                                                       FESO.sub.4. 7H.sub.2 O                                                                      1-1000 uM                                                       SeO.sub.2      1-100 uM                                                       NiCl.sub.2    1-1000 uM                                                       ______________________________________                                    

A control culture with no added nutrients was maintained for comparison.Three replicates of each experiment were conducted. The cultures weremaintained in the incubator at 37° C. Solids and gas concentrations weremonitored as a function of time for a period of 15-17 days. The same sixparameters were evaluated as in Example 1.

                  TABLE 11                                                        ______________________________________                                        Results of NiCl.sub.2 Amended Cultures                                        (after 15 days - all data are average of 3 replicates)                                             Vol-                                                             Dry          atile   Gas Production (ml)                              Amend-    Solids  Ash    Solids                                                                              Mea-  Calculated                               ment   pH     g/L     g/L  g/L   sured V.S. Methane                           ______________________________________                                        Begin- 7.49   11.860  3.348                                                                              8.512 0     0    0                                 ning                                                                          Control                                                                              7.71   9.191   3.036                                                                              6.155 59.7  59.1 63.2                                1 uM 7.72   9.283   3.077                                                                              6.206 62.7  57.6 61.5                               10 uM 7.70   9.301   3.055                                                                              6.246 68.3  56.5 61.1                               100 uM                                                                              7.72   9.328   3.049                                                                              6.279 68.7  55.6 60.2                              1000 uM                                                                              7.70   9.483   2.895                                                                              6.588 62.3  46.8 54.3                              ______________________________________                                    

                  TABLE 12                                                        ______________________________________                                        Methane Production from NiCl.sub.2 Amended Cultures                                    Methane Production (10.sup.-3 g mole/L)                                       Day                                                                  Amendment  1        3      5      9    15                                     ______________________________________                                        Control    4.589    7.532  8.988  9.849                                                                              10.794                                   1 uM     4.647    7.519  9.011  9.954                                                                              10.602                                  10 uM     4.611    7.523  9.093  9.906                                                                              10.609                                  100 uM    4.597    7.415  8.940  9.702                                                                              10.513                                 1000 uM    3.409    6.461  8.165  9.142                                                                              10.086                                 ______________________________________                                    

                  TABLE 13                                                        ______________________________________                                        Results of FeSO.sub.4 Amended Cultures                                        (after 15 days - all data are average of 3 replicates)                                             Vol-                                                             Dry          atile   Gas Production (ml)                              Amend-    Solids  Ash    Solids                                                                              Mea-  Calculated                               ment   pH     g/L     g/L  g/L   sured V.S. Methane                           ______________________________________                                        Begin- 7.62   7.696   2.391                                                                              5.305 0     0    0                                 ning                                                                          Control                                                                              7.80   6.217   2.157                                                                              4.060 43.3  30.4 34.6                               10 uM 7.77   6.319   2.199                                                                              4.120 45.3  28.7 34.1                               100 uM                                                                              7.80   6.273   2.193                                                                              4.080 41.7  29.9 34.6                              1000 uM                                                                              7.78   6.500   2.340                                                                              4.160 41.8  27.6 31.9                              ______________________________________                                    

                  TABLE 14                                                        ______________________________________                                        Methane Production from FeSO.sub.4 Amended Cultures                                    Methane Production (10.sup.-3 mole/L)                                         Day                                                                  Amendment  1        3      7      10   14                                     ______________________________________                                        Control    3.142    5.227  6.178  6.504                                                                              7.270                                   10 uM     3.182    5.274  6.138  6.524                                                                              7.264                                   100 uM    3.273    5.266  6.073  6.447                                                                              7.293                                  1000 uM    3.169    4.971  5.750  6.077                                                                              6.852                                  ______________________________________                                    

                  TABLE 15                                                        ______________________________________                                        Results of FeCl.sub.2 Amended Cultures                                        (after 15 days - all data are average of 3 replicates)                                             Vol-                                                             Dry          atile   Gas Production (ml)                              Amend-    Solids  Ash    Solids                                                                              Mea-  Calculated                               ment   pH     g/L     g/L  g/L   sured V.S. Methane                           ______________________________________                                        Begin- 7.97   8.824   2.592                                                                              6.232 0     0    0                                 ning                                                                          Control                                                                              7.78   6.847   2.353                                                                              4.494 37.0  43.6 44.6                               10 uM 7.77   7.056   2.403                                                                              4.653 48.3  39.1 42.7                               100 uM                                                                              7.78   6.703   2.204                                                                              4.499 46.7  43.5 44.1                              1000 uM                                                                              7.72   7.108   2.457                                                                              4.651 47.3  39.1 41.2                              5000 uM                                                                              7.58   7.912   2.769                                                                              5.143 44.0  25.1 35.5                              ______________________________________                                    

                  TABLE 16                                                        ______________________________________                                        Methane Production from FeCl.sub.2 Amended Cultures                                    Methane Production (10.sup.-3 mole/L)                                         Day                                                                  Amendment  1        4      8      11   17                                     ______________________________________                                        Control    3.352    5.996  7.473  7.644                                                                              8.460                                   10 uM     3.378    6.077  7.437  7.728                                                                              8.386                                   100 uM    3.282    5.966  7.467  7.925                                                                              8.387                                  1000 uM    3.277    5.791  7.209  7.487                                                                              8.091                                  5000 uM    3.042    5.518  6.876  7.236                                                                              7.784                                  ______________________________________                                    

                  TABLE 17                                                        ______________________________________                                        Results of SeO.sub.2 Amended Cultures                                         (after 15 days - all data are average of 3 replicates)                                             Vol-                                                             Dry          atile   Gas Production (ml)                              Amend-    Solids  Ash    Solids                                                                              Mea-  Calculated                               ment   pH     g/L     g/L  g/L   sured V.S. Methane                           ______________________________________                                        Begin- 7.97   14.172  4.428                                                                              9.744 0     0    0                                 ning                                                                          Control                                                                              7.77   11.009  3.931                                                                              7.078 70.2  66.7 62.8                               1 uM  7.81   11.103  3.923                                                                              7.180 72.0  63.8 63.5                               10 uM 7.94   11.121  3.939                                                                              7.182 72.7  63.8 61.5                              100 uM 7.96   11.144  3.979                                                                              7.165 72.7  64.2 59.8                              ______________________________________                                    

                  TABLE 18                                                        ______________________________________                                        Methane Production from SeO.sub.2 Amended Cultures                                     Methane Production (10.sup.-3 mole/L)                                         Day                                                                  Amendment  1        3      5      11   15                                     ______________________________________                                        Control    3.765    6.736  7.780  9.510                                                                              10.223                                  1 μM   3.893    6.753  7.805  9.668                                                                              10.528                                  10 μM  3.966    6.849  7.891  9.500                                                                              10.201                                 100 μM  3.846    6.605  7.607  9.327                                                                               9.888                                 ______________________________________                                    

The consistency of the data in Tables 11-18 was reviewed on the basis ofvolatile solids conversion and gas and methane production. For example,Table 11 shows that the volatile solids were reduced from 8.512 g/L to6.155 for the NiCl₂ control culture. The volatile solids destroyedshould produce 59.1 ml of total gas, which compares closely with the59.7 ml measured. The gas volume computed from the methane produced inTable 12 is 63.2 ml, close to the value computed from volatile solidsand measured.

EXAMPLE 3 Addition of Chelating Agents and Nutrients

These experiments were conducted in the same manner as the chelating andnutrient experiments described in Examples 1 and 2. In theseexperiments, FeSO₄ was added alone and along with either CA or CDTA.

The results are presented in Tables 19-20. The same six parameters wereevaluated and the data are in the same form as previously described.

                  TABLE 19                                                        ______________________________________                                        Results of 1000 uM FeSO.sub.4 and Chelating Agent Amended Sludge              (After 17 days - all data are average of 3 replicates)                                             Vol-                                                             Dry          atile   Gas Production (ml)                              Amend-        Solids  Ash  Solids      Calculated                             ment   pH     g/L     g/L  g/L   Meas. V.S. Methane                           ______________________________________                                        Begin- 7.39   16.368  5.239                                                                              11.129                                                                              0     0    0                                 ning                                                                          Control                                                                              7.62   12.063  4.572                                                                              7.491 85.7  98.2 105.1                             1000 uM                                                                              7.59   12.516  4.804                                                                              7.712 74.7  86.8 94.6                              FeSO                                                                          1000 uM                                                                              7.63   12.597  4.865                                                                              7.732 88.7  86.2 97.7                              FeSO                                                                          +20 uM                                                                        CACA                                                                          1000 uM                                                                              7.74   12.571  4.849                                                                              7.722 95.7  86.5 101.2                             FeSO                                                                          +20 uM                                                                        CDTA                                                                          ______________________________________                                    

                  TABLE 20                                                        ______________________________________                                        Methane Production from 1000 uM FeSO.sub.4 and                                Chelating Agent Amended Sludge                                                            Methane Production (10.sup.-3 mole/L)                                         Day                                                               Amendment     1      4         9     17                                       ______________________________________                                        Control       6.842  10.778    13.705                                                                              14.318                                   1000 uM FeSO  6.585  10.410    13.429                                                                              13.688                                   1000 uM FeSO  6.755  10.813    13.777                                                                              14.181                                   +20 uM CA                                                                     1000 uM FeSO  6.833  10.978    13.893                                                                              14.665                                   +20 uM CDTA                                                                   ______________________________________                                    

EXAMPLE 4 Addition of Methanogens--Batch Experiments

Sludge was introduced into serum bottles as described in Example 1. Purecultures of methanogens grown in a mineral salts medium without richorganics were used for injection into the bottles of sludge. Thefollowing methanogens were used:

Methanosarcina barkeri (MB)

Methanobrevibacter smithii (MS)

Methanobacterium formicium (MF)

The methanogens used in this study were grown in 1 liter bottlescontaining biphasic medium (250 ml of agar and 80 ml of liquid medium).The headspace contained 80% H₂ :20% CO₂ at 20 psi. This method producedcells at 20-25 grams per liter in four days. These cells were theninjected into the digester sludge anaerobically using syringes.

These organisms were inoculated singly and in combination. A quantity of2 ml of the pure culture containing 20-25 g/L methanogens was added to28 ml of sludge in the serum bottle.

The data from these experiments are shown in Tables 21 and 22.

                  TABLE 21                                                        ______________________________________                                        Results of Methanogen Amended Sludge                                          (After 18 days - all data are average of three replicates)                                         Vol-                                                             Dry          atile   Gas Production (ml)                              Amend-    Solids  Ash    Solids      Calculated                               ment   H      g/L     g/L  g/L   Meas. V.S. Methane                           ______________________________________                                        Begin- 8.02   16.128  5.276                                                                              10.852                                                                              0     0    0                                 ning                                                                          Control                                                                              7.81   13.254  4.871                                                                              8.383 63.0  49.7 56.1                              MF     7.89   12.991  4.856                                                                              8.135 70.3  56.8 62.2                              MB     7.84   13.064  4.856                                                                              8.208 78    54.7 64.4                              MS     7.83   12.946  4.826                                                                              8.120 67.3  57.2 62.1                              MF +   7.91   12.851  4.813                                                                              8.038 68.3  59.6 63.5                              MB                                                                            MF +   7.94   12.827  4.749                                                                              8.078 68.3  58.4 61.2                              MS                                                                            MB +   7.84   12.689  4.727                                                                              7.962 66.0  61.7 64.3                              MS                                                                            MF +   7.75   12.624  4.764                                                                              7.860 62.3  64.7 65.4                              MB +                                                                          MS                                                                            ______________________________________                                    

                  TABLE 22                                                        ______________________________________                                        Methane Production from Cultures Amended with Methanogens                                 Methane Production (10.sup.-3 g mole/L)                                       Day                                                               Amendment     1      3        5    10     18                                  ______________________________________                                        Control       4.05   7.33     8.71 9.30   10.20                               MF            4.85   7.78     9.18 9.75   10.79                               MB            5.21   8.01     9.66 10.08  11.32                               MS            4.91   7.78     9.34 9.72   10.73                               MF + MB       4.98   7.76     9.22 9.73   10.81                               MF + MS       4.92   7.74     9.15 9.65   10.49                               MB + MS       5.02   7.95     9.34 9.95   10.79                               MF + MB + MS  4.97   7.79     9.22 9.85   10.78                               ______________________________________                                    

EXAMPLE 5 Addition of Methanogens--Continuous Experiments, Start-up

Semi-continuous reactors were used to evaluate the enhancement of adigester start-up with the addition of methanogens. Five-liter Plexiglasreactors, with a three-liter liquid volume, as illustrated in FIG. 3,were utilized. The reactors were equipped with continuous mechanicalagitators. Substrate was introduced daily through a valve and entranceport below the liquid level. Gas was discharged through an opening atthe top and collected in polyethylene bags for measurement in a wet testmeter. The reactors were incubated at 37° C.

Thickened sludge (feed material to the digesters from the Springdale,Arkansas wastewater treatment plant) was used as substrate for theseexperiments. The characteristics of the sludge were as follows:

    ______________________________________                                        pH                   5.32                                                     Total Solids         47.969  g/L                                              Ash                  8.019   g/L                                              Volatile Solids      39.95   g/L                                              ______________________________________                                    

Two reactors were started simultaneously. Both reactors were adjusted topH 7 and were inoculated with 500 ml of active anaerobic digester sludgeand 2.4 L distilled water. One reactor was also amended with 10 uMcitric acid and was inoculated with 250 ml of a pure culture ofMethanosarcina barkeri (MB) which had been grown to a concentration ofapproximately 20 g/L.

Both reactors were operated the same, being fed thickened sludge on afive-day retention time. The pH was adjusted daily with sodiumbicarbonate. The reactors were agitated at approximately 100 rpm. Themethanogen amended reactor was also fed citric acid daily to maintain aconcentration of 10 uM. Gas volumes and concentrations were measureddaily. The methane production and improvements are shown on Table 23.

                  TABLE 23                                                        ______________________________________                                        Methane Production from MB Amended Culture                                    Methane production (L/wk)                                                     Week    Control    MB Amended Improvement %                                   ______________________________________                                        1        9.754     12.325     26.4                                            2       13.553     17.529     29.3                                            3       15.192     20.099     32.3                                            4       14.652     15.908     8.6                                             ______________________________________                                    

The importance of these results is apparent in more rapid starts ofdigesters. For the first two weeks, the amended culture reached the samegas production level as the control within 3-4 days less time. However,for the last two weeks, the same gas production was achieved 4-6 daysquicker. Therefore, digester start-up times could be shortened by about25 percent, or perhaps more with a longer retention time.

More importantly, the increased rate of methane production is sustainedeven after four weeks. This indicates that such an improvement might besustained over an extended period, perhaps with periodic addition ofmethanogens.

EXAMPLE 6 Addition of Methanogens--Continuous Experiments/Recovery

Additional continuous experiments were conducted to determine the effectwhich the addition of methanogens would have on the recovery of an upsetdigester. The two reactors used for the start-up experiments describedin Example 5 were shocked by depressing the pH to 4.5 for 48 hours. Bothreactors were then adjusted to pH 7 and one reactor was amended byaddition of 250 ml of Methanosarcina barkeri and 10 uM citric acid. Thereactors were then operated on a 10-day retention time over a two-weekperiod.

The gas production rates for each two day period are given in Table 24for the two reactors. Gas compositions were around 60 percent methane.FIG. 5 shows the cumulative gas production over the two week period.After a period of two days, the amended culture began producing gas at arate similar to that of a normal digester. The rate continued to improveand, after two weeks, the gas production had nearly doubled the control.The marked difference between the start-up reactor and the shockedreactor can be attributed to the longer retention time and fact that ahigher methanogen population had been established prior to the shock.

                  TABLE 24                                                        ______________________________________                                        Total Gas Production During Recovery of Upset Digester                        Gas Production (L/wk)                                                         Control         MB Amended Improvement                                        ______________________________________                                        1/2     0.947       1.647      74                                             3/4     2.124       3.123      47                                             5/6     1.320       2.532      92                                             7/8     1.150       2.351      104                                             9/10   0.761       2.407      216                                            11/12   1.678       2.561      53                                             13/14   2.999       4.571      52                                             ______________________________________                                    

EXAMPLE 7 Chelation Optimizing Experiments--Continuous Culture

Two-liter reactors, with IL of liquid culture volume were used in thisexperiment. All the reactors were incubated inside a cabinet maintainedat 38°0 C. Autoclaved sludge was fed daily in the quantity required tomaintain the desired retention time. An equal amount of effluent waswithdrawn. The sludge was obtained periodically from-the Springdale,Arkansas Waste Treatment facility. The gas produced was collected inpolyethylene bags through quick connect fittings and was measured onceevery three days by a water displacement facility. Control reactors wereoperated under identical conditions for comparison. Gas composition andthe total solids, volatile solids, and ash content of the effluent weremeasured periodically.

Three chelating agents, CA, NTA, and EDTA, were utilized inconcentrations from 1-500 uM. The chelating agent studies included 10and 100 uM concentrations at 10, 15 and 25 day retention times and a 500uM concentration at a 25 day retention time. The concentrations (uM) ofchelator relate to the sludge additions only. The results of thesestudies are summarized in Table 25 and FIGS. 6 and 7. The results aregiven as the percentage of improvement over the control reactor. Theaverages stated are ±10 percent in the 95 percent confidence interval.

                  TABLE 25                                                        ______________________________________                                        Average Gas Production of Chelating Agent Test and                            Percent Improvement over the Control.                                                Retention Time                                                                10 days   15 days     25 days                                                 L/day Imprv.  L/day   Imprv.                                                                              L/day Imprv.                               ______________________________________                                        A.  10 uM                                                                         Conc.                                                                         Control  1.934         1.224       0.571                                      EDTA     2.005   3.7%  1.532 25.2% 0.754 32.0%                                CA       2.111   9.2%  1.414 15.6% 0.786 37.7%                                NTA                    1.391 13.6%                                        B.  100 uM                                                                        Conc.                                                                         Control  1.583         1.297       0.533                                      EDTA     1.622   2.5%  1.646 26.9% 0.711 33.5%                                CA       1.849   16.8% 1.480 14.1% 0.683 28.1%                                NTA      1.690   6.8%  1.457 12.3% 0.663 24.5%                            C.  500 uM                                                                        Conc.                                                                         Control                            0.583                                      EDTA                               0.892 53.0%                                Control                            0.567                                      CA                                 0.838 47.8%                            ______________________________________                                    

FIGS. 6 and 7 show the improvement in gas production vs. dosage of EDTAand CA amendment, respectively. As shown in these figures, theimprovement is more sensitive to the retention time than to theamendment concentration. As expected, the longer the retention time, thegreater the improvement. At low retention time (higher flow rates),adequate dissolved nutrients are supplied for microbial growth. However,at longer retention times, solubilized nutrients required for maximumgrowth are insufficient without the use of chelating agents. Also, asnoted in Table 25, larger quantities of gas are produced at shorterretention times; therefore, the percentage improvement is less for thesame increase in gas production. However, more chelator is added at thehigher flow rates.

FIG. 8 shows the combined (average) improvement for both CA and EDTA.The greatest improvement is achieved with a 10 uM dosage. Above thislevel, the improvement shows a constant slope. The amount of methaneproduced from the chelator added at 500 uM could account for only a onepercent or less improvement. Therefore, some enhancement is achievedwith dosages above 10 uM, although the enhancement is only about 3percent per 100 uM added.

The combined gas production and improvement for both EDTA and CAamendment, with different dosages, was plotted vs. retention time inFIG. 9. The improvement is proportional to the retention time, with aslope of 2.1 percent per day. Most commercial digesters operate at 25day-retention times or longer. Therefore, even greater percentageimprovements can be expected with chelation amendment. FIG. 9 also showsthe expected gas production rates (per liter of liquid volume). Thesedata give a negative slope with increasing retention time. Because theamount of volatile solids fed at long retentions is less than the amountfed at short retentions, the quantity of gas produced decreases asretention time increases.

EXAMPLE 8 Annual Savings Estimates

The base case in this study is a 2.4 mill-ion liter digester, with theassumption that 60 percent of the gas produced is methane and methanehas a value of $3.0 per thousand cubic feet (MCF). The normal gasproduction is based on 75 percent of the average gas production found inTable 25. The gas production rates in Table 25 were obtained under ideallaboratory conditions and 75 percent of these values is more realistic.Thus, the normal annual methane production is 24,508, 17,569, and 7,856MCF/yr for 10, 15, and 25 days retention time, respectively. Theincreased methane production by the amendment is found from FIGS. 6 and7. The annual chelating agent cost is estimated as the sum of the costof the initial amendment and the further additions to maintain thedosage concentration for one year. The annual usage of chelating agentis listed in Table 26. The current unit costs of EDTA and CA are$0.803/Kg and $1,837/Kg, respectively. Therefore, the net benefit of theamendment can be found by subtracting the chelating agent cost from thevalue of the added methane production. In addition, cost benefits resultfrom a reduction in sludge quantity to be disposed, which could exceedthe increased methane value. Perhaps more importantly, the need forincreased physical capacity of existing facilities can be delayed byincreasing capacities with chelation.

                  TABLE 26                                                        ______________________________________                                        Annual Chelating Agent Required Amount                                                  Chelator Concentration*                                             Retention Time                                                                            10 uM       100 uM  500 uM                                        ______________________________________                                        10 days     900         9,000   45,000                                        15 days     608         6,179   30,396                                        25 days     375         3,744   18,720                                        ______________________________________                                         *values in gmoles/yr                                                     

EXAMPLE 9 Extended Continuous Methanogen Experiments

Methanosarcina barkeri was added to 2L bench scale digesters (asdescribed in Example 7) in varying quantities and on differentschedules. Continuous experiments were initiated using an inoculum ofMethanosarcina barkeri at the level of 1 g/L to determine theenhancement of pure culture methanogens. Two reactors were started withfresh sludge at a 25-day retention time. When the performance of bothreactors were parallel, one reactor was inoculated with 75 ml ofMethanosarcina barkeri at a concentration of 12.01 g/l to bring thecontents of the reactor to a concentration of about 1 g/L. The otherreactor served as a control. These reactors were operated for anextended period of five months.

The gas production of these reactors is listed in Table 27 and plottedin FIGS. 12 and 13. The performance steadily improved, with gasproduction exceeding the control by 9 percent the first month, 22percent the second month, 32 percent the third month, 38 percent thefourth month, and 41 percent the fifth month. The cumulative improvementaveraged 35 percent. The data indicated that during the last month, thecontinued improvement of the amended reactor over the control waslargely due to the slower recovery of the control reactor to variationsin sludge quality. After 120 days, the sludge quality changed, and thegas production in the control reactor declined sharply. The amendedreactor, however, showed no decline at 120 days, indicating excellentstability. Methane concentrations averaged about 66 percent of total gasproduction and pH remained normal over the five-month period. Theimproved performance of the amended reactor appears to be permanent atthe 25-day retention time, with no evidence of a decline in gasproduction after five months.

                                      TABLE 27                                    __________________________________________________________________________    Gas Production and pH of 1 g/L Methanogen Test                                METHANOGEN:                                                                              CONTROL        METHANOSARCINA                                      CONCENTRATION:                                                                           0.0 G/L        1.0 G/L                                             RETENTION TIME:                                                                          25.0 DAYS      25.0 DAYS                                           DATE       V L/DAY                                                                             ACC. V L                                                                            pH V L/DAY                                                                             ACC. V L                                                                            pH                                      __________________________________________________________________________     6 18 87   0.504  1.511                                                                              7.31                                                                             0.668  2.005                                                                              7.33                                     6 21 87   0.741  3.735                                                                              7.34                                                                             0.702  4.111                                                                              7.36                                     6 24 87   0.480  5.176                                                                              7.38                                                                             0.675  6.135                                                                              7.32                                     6 27 87   0.650  7.127                                                                              7.38                                                                             0.615  7.981                                                                              7.37                                     6 30 87   0.582  8.875                                                                              7.49                                                                             0.593  9.760                                                                              7.49                                     7  3 86   0.469 10.282                                                                              7.45                                                                             0.576 11.487                                                                              7.48                                     7  6 87   0.580 12.022                                                                              7.46                                                                             0.650 13.437                                                                              7.40                                     7  9 87   0.566 13.719                                                                              7.46                                                                             0.555 15.102                                                                              7.40                                     7 12 87   0.553 15.378                                                                              7.33                                                                             0.569 16.808                                                                              7.34                                     7 15 87   0.530 15.968                                                                              7.33                                                                             0.568 18.511                                                                              7.36                                     7 18 87   0.377 18.099                                                                              7.44                                                                             0.549 20.159                                                                              7.42                                     7 21 87   0.248 18.841                                                                              7.48                                                                             0.688 22.222                                                                              7.47                                     7 24 87   0.096 19.129                                                                              7.47                                                                             0.659 24.198                                                                              7.45                                     7 27 87   0.099 19.425                                                                              7.34                                                                             0.599 25.996                                                                              7.28                                     7 30 87   0.493 20.905                                                                              7.29                                                                             0.646 27.935                                                                              7.30                                     8  2 87   0.447 22.245                                                                              7.31                                                                             0.726 30.113                                                                              7.30                                     8  5 87   0.491 23.718                                                                              7.31                                                                             0.668 32.117                                                                              7.30                                     8  8 87   0.576 24.445                                                                              7.29                                                                             0.659 34.094                                                                              7.20                                     8 11 87   0.610 27.275                                                                              7.30                                                                             0.739 36.311                                                                              7.28                                     8 14 87   0.620 29.136                                                                              7.28                                                                             0.626 38.189                                                                              7.30                                     8 17 87   0.665 31.130                                                                              7.08                                                                             0.526 39.767                                                                              7.04                                     8 20 87   0.670 33.142                                                                              7.28                                                                             0.713 41.905                                                                              7.31                                     8 23 87   0.590 34.913                                                                              7.38                                                                             0.836 44.414                                                                              7.35                                     8 26 87   0.670 36.923                                                                              7.29                                                                             0.763 46.702                                                                              7.27                                     8 29 87   0.592 38.700                                                                              7.31                                                                             0.860 49.281                                                                              7.30                                     9  1 87   0.461 40.084                                                                              7.33                                                                             0.672 51.296                                                                              7.39                                     9  4 87   0.312 31.020                                                                              7.39                                                                             0.504 52.809                                                                              7.37                                     9  7 87   0.102 31.325                                                                              7.40                                                                             0.557 54.479                                                                              7.41                                     9 10 87   0.392 42.503                                                                              7.43                                                                             0.477 55.910                                                                              7.45                                     9 13 87   0.404 43.715                                                                              7.40                                                                             0.516 57.457                                                                              7.38                                     9 16 87   0.364 44.807                                                                              7.44                                                                             0.486 58.914                                                                              7.44                                     9 19 87   0.405 46.022                                                                              7.45                                                                             0.492 60.390                                                                              7.45                                     9 22 87   0.387 47.182                                                                              7.35                                                                             0.474 61.813                                                                              7.36                                     9 25 87   0.461 48.565                                                                              7.32                                                                             0.524 63.385                                                                              7.35                                     9 28 87   0.446 49.903                                                                              7.24                                                                             0.540 65.005                                                                              7.32                                    10  1 87   0.502 51.410                                                                              7.28                                                                             0.577 66.736                                                                              7.33                                    10  4 87   0.471 42.825                                                                              7.30                                                                             0.667 68.739                                                                              7.34                                    10  7 87   0.462 54.211                                                                              7.35                                                                             0.733 70.938                                                                              7.36                                    10 10 87   0.577 55.941                                                                              7.41                                                                             0.757 73.208                                                                              7.44                                    10 13 87   0.425 57.215                                                                              7.24                                                                             0.743 75.438                                                                              7.40                                    10 16 87   0.431 58.509                                                                              7.40                                                                             0.811 77.872                                                                              7.40                                    10 19 87   0.470 59.919                                                                              7.40                                                                             0.695 79.957                                                                              7.42                                    10 22 87   0.440 61.240                                                                              7.37                                                                             0.627 81.839                                                                              7.44                                    10 25 87   0.506 62.757                                                                              7.37                                                                             0.732 84.935                                                                              7.41                                    10 28 87   0.634 64.658                                                                              7.39                                                                             0.733 86.235                                                                              7.41                                    10 31 87   0.655 66.623                                                                              7.39                                                                             0.861 88.818                                                                              7.41                                    11  3 87   0.566 68.322                                                                              7.37                                                                             0.875 91.444                                                                              7.36                                    11  6 87   0.556 69.991                                                                              7.33                                                                             0.903 94.153                                                                              7.34                                    __________________________________________________________________________     NOTE: V: GAS PRODUCTION RATE IN STP L/DAY                                     ACC. V: ACCUMULATED GAS PRODUCTION IN STP L                                   CONT: CONTROL                                                            

An additional 1 g/L of Methanosarcina barkeri was added to themethanogen reactor after the five-month period. The re-amended reactorwas operated for another four months. The performance of this reactorand control are given in Table 28 and FIGS. 14 and 15. The average gasproduction was further improved to a level of 50% following the secondinoculation. This level of improvement was sustained for the four monthperiod and it was concluded that the enhancement was permanent, or atleast long term. The gas production averaged 0.899 L/day from themethanogen reactor and 0.567 L/day for the control. Thus, the additionof methanogens resulted in a 59 percent improvement.

                                      TABLE 28                                    __________________________________________________________________________    Gas Production and pH of 2 g/L Methanogen Test                                METHANOGEN:                                                                              CONTROL        METHANOSARCINA                                      CONCENTRATION:                                                                           0.0 G/L        1.0 G/L                                             RETENTION TIME:                                                                          25.0 DAYS      25.0 DAYS                                           DATE       V L/DAY                                                                             ACC. V L                                                                            pH V L/DAY                                                                             ACC. V L                                                                            pH                                      __________________________________________________________________________    11 15 87   0.571  1.713                                                                              7.39                                                                             0.882  2.646                                                                              7.38                                    11 18 87   0.516  3.262                                                                              7.43                                                                             0.800  5.044                                                                              7.42                                    11 21 87   0.516  4.810                                                                              7.45                                                                             0.765  7.341                                                                              7.45                                    11 24 87   0.644  6.743                                                                              7.45                                                                             0.926 10.118                                                                              7.41                                    11 27 87   0.609  8.569                                                                              7.48                                                                             0.758 12.392                                                                              7.48                                    11 30 87   0.655 10.533                                                                              7.30                                                                             0.851 14.945                                                                              7.34                                    12  3 87   0.659 12.509                                                                              7.34                                                                             0.844 17.477                                                                              7.34                                    12  6 87   0.538 14.122                                                                              7.47                                                                             0.745 19.712                                                                              7.46                                    12  9 87   0.614 15.963                                                                              7.43                                                                             0.813 22.151                                                                              7.41                                    12 12 87   0.553 17.621                                                                              7.42                                                                             0.836 24.660                                                                              7.39                                    12 15 87   0.475 19.046                                                                              7.41                                                                             0.835 27.164                                                                              7.41                                    12 18 87   0.441 20.371                                                                              7.38                                                                             0.623 29.035                                                                              7.40                                    12 21 87   0.454 21.733                                                                              7.43                                                                             0.727 31.216                                                                              7.41                                    12 24 87   0.497 23.224                                                                              7.45                                                                             0.833 33.716                                                                              7.43                                    12 27 87   0.633 25.122                                                                              7.41                                                                             0.722 35.882                                                                              7.39                                    12 31 87   0.624 26.994                                                                              7.39                                                                             0.807 38.303                                                                              7.41                                     1  2 88   0.500 28.495                                                                              7.39                                                                             0.812 40.739                                                                              7.36                                     1  5 88   0.404 29.706                                                                              7.38                                                                             0.621 42.602                                                                              7.38                                     1  8 88   0.509 31.232                                                                              7.30                                                                             0.726 44.779                                                                              7.27                                     1 11 88   0.473 32.650                                                                              7.38                                                                             1.066 47.977                                                                              7.37                                     1 14 88   0.680 34.691                                                                              7.35                                                                             0.918 50.732                                                                              7.31                                     1 17 88   0.638 36.604                                                                              7.39                                                                             0.692 52.802                                                                              7.38                                     1 20 88   0.752 38.862                                                                              7.39                                                                             1.044 55.941                                                                              7.38                                     1 23 88   0.497 40.352                                                                              7.29                                                                             1.189 59.508                                                                              7.35                                     1 26 88   0.526 41.931                                                                              7.48                                                                             1.041 62.632                                                                              7.45                                     1 29 88   0.764 44.223                                                                              7.44                                                                             1.062 65.816                                                                              7.43                                     2  1 88   0.680 46.262                                                                              7.47                                                                             1.157 69.287                                                                              7.48                                     2  4 88   0.569 47.969                                                                              7.41                                                                             1.029 72.373                                                                              7.42                                     2  7 88   0.630 49.859                                                                              7.40                                                                             1.081 75.615                                                                              7.36                                     2 10 88   0.586 51.618                                                                              7.46                                                                             0.975 78.539                                                                              7.46                                     2 13 88   0.590 53.388                                                                              7.44                                                                             1.199 83.136                                                                              7.45                                     2 16 88   0.645 55.324                                                                              7.40                                                                             1.045 85.272                                                                              7.38                                     2 19 88   0.625 57.199                                                                              7.48                                                                             0.927 88.054                                                                              7.48                                     2 22 88   0.471 58.613                                                                              7.38                                                                             0.812 90.489                                                                              7.42                                     2 25 88   0.643 60.543                                                                              7.51                                                                             0.841 92.012                                                                              7.52                                     2 28 88   0.547 62.184                                                                              7.40                                                                             0.847 95.553                                                                              7.38                                     3  2 88   0.537 63.795                                                                              7.41                                                                             0.838 98.067                                                                              7.40                                     3  5 88   0.477 65.226                                                                              7.45                                                                             0.872 100.681                                                                             7.44                                     3  8 88   0.603 67.036                                                                              7.48                                                                             0.780 103.022                                                                             7.50                                    __________________________________________________________________________     NOTE: V: GAS PRODUCTION RATE IN STP L/DAY                                     ACC. V: ACCUMULATED GAS PRODUCTION IN STP L                                   CONT: CONTROL                                                            

EXAMPLE 10 Recovery of Methanogen Amended Reactor From Upset

These experiments were conducted as described in Example 8. Both thecontrol and methanogen reactors were upset by adding sulfuric acid toreduce the pH to 4.0 for one day after which the pH was returned to 7.0by adding 10% sodium bicarbonate. Table 29 and FIGS. 16 and 17 show theperformance of these reactors following the upset. The gas productionfor both reactors was severely inhibited during the first three weeks.However, the gas production for both reactors quickly recovered and theproduction levels were increased to about twice the pre-upset levels.The methane composition for both reactors reduced to 45% during theinhibited period, but returned to the 63% level when the gas productionrecovered. The unexpected high gas production lasted about two monthswith practically no difference in the production rates between the tworeactors. The gas production of the control reactor then started todecline after two months. The gas production of the amended reactor didnot decline and the improvement was about 60% for the remainder of theexperiment.

                                      TABLE 29                                    __________________________________________________________________________    Gas Production and pH for the Upset Methanogen Reactor                        METHANOGEN:                                                                              CONTROL        METHANOSARCINA                                      CONCENTRATION:                                                                           0.0 G/L        3 G/L                                               RETENTION TIME:                                                                          25.0 DAYS      25.0 DAYS                                           DATE       V L/DAY                                                                             ACC. V L                                                                            pH V L/DAY                                                                             ACC. V L                                                                            pH                                      __________________________________________________________________________    3 18 88    0.152  0.455                                                                              6.95                                                                             0.282  0.563                                                                              7.01                                    3 20 88    0.269  1.144                                                                              7.06                                                                             0.355  1.555                                                                              7.13                                    3 23 88    0.164  1.636                                                                              7.07                                                                             0.202  2.162                                                                              7.16                                    3 26 88    0.137  2.047                                                                              7.23                                                                             0.207  2.783                                                                              7.28                                    3 29 88    0.147  2.488                                                                              7.32                                                                             0.207  3.404                                                                              7.35                                    4  1 88    0.219  3.146                                                                              7.34                                                                             0.291  4.275                                                                              7.34                                    4  4 88    0.186  3.704                                                                              7.33                                                                             0.194  4.859                                                                              7.36                                    4  7 88    0.170  4.214                                                                              7.37                                                                             0.192  5.434                                                                              7.37                                    4 13 88    0.167  4.716                                                                              7.34                                                                             0.237  6.144                                                                              7.42                                    4 16 88    0.405  5.931                                                                              7.32                                                                             0.478  7.579                                                                              7.31                                    4 19 88    0.585  7.686                                                                              7.20                                                                             0.662  9.564                                                                              7.24                                    4 22 88    0.684  9.739                                                                              7.36                                                                             0.856 12.134                                                                              7.31                                    4 26 88    0.846 12.276                                                                              7.34                                                                             0.977 15.064                                                                              7.36                                    4 28 88    1.040 15.396                                                                              7.39                                                                             1.112 18.399                                                                              7.42                                    5  1 88    1.264 19.189                                                                              7.66                                                                             1.351 22.452                                                                              7.66                                    5  4 88    1.173 22.707                                                                              7.59                                                                             1.312 26.386                                                                              7.65                                    5  8 88    1.106 26.026                                                                              7.64                                                                             1.682 31.433                                                                              7.68                                    5  8 88    0.995 29.012                                                                              7.62                                                                             1.060 34.614                                                                              7.64                                    5 13 88    1.128 32.395                                                                              7.61                                                                             1.270 38.422                                                                              7.62                                    5 16 88    1.073 35.612                                                                              7.56                                                                             0.958 41.297                                                                              7.57                                    5 19 88    0.975 38.539                                                                              7.57                                                                             0.961 44.181                                                                              7.66                                    5 22 88    1.090 41.809                                                                              7.61                                                                             1.033 47.281                                                                              7.69                                    5 25 88    1.006 44.825                                                                              7.53                                                                             1.265 51.076                                                                              7.56                                    5 28 88    1.062 48.010                                                                              7.57                                                                             0.928 53.858                                                                              7.59                                    5 31 88    1.285 51.866                                                                              7.45                                                                             1.225 57.533                                                                              7.48                                    6  3 88    1.275 55.690                                                                              7.56                                                                             1.280 61.372                                                                              7.62                                    6  6 88    1.329 59.677                                                                              7.58                                                                             1.096 64.660                                                                              7.63                                    6  9 88    0.786 62.034                                                                              7.56                                                                             1.265 68.455                                                                              7.63                                    6 12 88    0.644 63.965                                                                              7.55                                                                             1.340 72.475                                                                              7.55                                    6 15 88    0.737 66.176                                                                              7.56                                                                             1.395 76.659                                                                              7.60                                    6 18 88    0.893 68.854                                                                              7.50                                                                             1.663 81.649                                                                              7.59                                    6 21 88    0.842 71.380                                                                              7.55                                                                             1.594 86.431                                                                              7.66                                    __________________________________________________________________________     NOTE: V: GAS PRODUCTION RATE IN STP L/DAY                                     ACC. V: ACCUMULATED GAS PRODUCTION IN STP L                                   CONT: CONTROL                                                            

The unexpected high gas production from the upset reactors may have beencaused by the use of sulfate. The sulfuric acid introduced is convertedto sulfate when the acid is neutralized. Sulfate enhances the growth ofsulfate reducing bacteria. There is recent evidence that some sulfatereducing bacteria, such as Desulfobacter nostaatei, utilize both H₂--CO₂ and acetate faster than methanogens. Therefore, the high gasproduction of both the reactors after the inhibition period may havebeen caused by the enhanced growth of sulfate reducing bacteria. Afterall the sulfate was displaced and consumed, the control reactor returnedto a normal production level. The amended reactor stayed at the higherlevel of enhanced performance.

EXAMPLE 11 Field Trials

Data to establish the normal operation of the treatment plant wereobtained prior to amendment. These base-line data were obtained for aone month period during the Summer 1987. Samples of the sludge enteringand leaving the two anaerobic digesters were taken daily and analyzedfor total solids and volatile solids. Data were also available fromroutine measurements by plant personnel. A comparison of results isshown in Table 30. Reasonable agreement is obtained, considering thedifferences in sampling times. The other information collected from theplant included the daily feed volumes and digester temperature.

                                      TABLE 30                                    __________________________________________________________________________    Sludge Characteristics at the Springdale, AR Wast Treatment Facility.                   Average Concentration (g/l) July 1987                                         Total Solids    Volatile Solids                                               Plant Personnel                                                                       Lab Personnel                                                                         Plant Personnel                                                                       Lab Personnel                               __________________________________________________________________________    Entering  23.89   20.25   17.49   16.69                                       Leaving Digester 1                                                                              15.06           10.26                                       Leaving Digester 2                                                                              15.85           10.79                                       __________________________________________________________________________

Variability in the sludge feed concentrations on a daily basis wasconsiderable (±60 percent). The effluent was somewhat more stable, butstill varied ±30 percent. Table 31 gives the monthly averages for thevolatile solids from the two digesters. The data show reasonableconsistency on a monthly basis, but considerable variability wasexperienced on a daily basis.

                  TABLE 31                                                        ______________________________________                                        Springdale Sludge Concentration in July and August 1987                              Volatile Solids Concentration (g/l)                                    Digester July         August  Combined                                        ______________________________________                                        1        10.33        9.64    9.88                                            2        10.74        11.14   10.99                                           ______________________________________                                    

CA AMENDMENT

Field experiments at the Springdale Treatment Plant were initiated onNov. 17, 1987. A 10 uM concentration of CA was added to Digester 2.Digester 1 was used as a control. The initial dosage of CA was 12.3 lbin water solution.

The feed rate of sludge varied considerably, from 30,000 to 100,000gallons per day. The average rate was 45-50,000 gallons per day, and theaverage rate of addition of CA was 0.8 lb/day to maintain 10 uM. Thesolution of CA was pumped into the sludge circulation line.

The temperature of Digester 2 steadily declined and after about onemonth, the temperature was down to 80° F. with a 15° F. differencebetween Digester 1 and 2. Because of the temperature control problems,the two digesters were operated at significantly different retentiontimes. Therefore, the addition of CA to Digester 2 was discontinued onJan. 4, 1988. The amendment of 10 uM CA was shifted to Digester 1 onJanuary 18. However, Digester 1 soon experienced a temperature reductionto about 88° F. There was a serious upset caused by overloading in earlyFebruary and the field experiments were concluded on Mar. 15, 1988.

A summary of the weekly performances of these two digesters is given inTables 32 and 33. Since the retention times and temperatures betweenthese two digesters were significantly different, the comparison of eachdigester with its own previous level of performance is probably moremeaningful.

It is generally agreed that the kinetics of anaerobic digestion can bedescribed by a first order reaction. The steady state equation of amixed reactor with first order kinetics is given by:

    k*RT=(C.sub.i -C.sub.o)/C.sub.o

where k is the reaction constant, RT is the retention time, C_(i) is thevolatile solids concentration of the feed, and C_(o) is the volatilesolids concentration of the leaving stream. Thus, a plot of (C_(i)-C_(o))/C_(o) vs RT should be linear, passing through the origin with aslope of k.

The plots of the (C_(i) -C_(o))/C_(o) vs RT for weekly average data ofeach reactor are shown in FIGS. 18 and 19. FIG. 18 describes theperformance of Digester 1. Linear relationships are shown for amendedand unamended periods. The slope (value of k) of the best fit for theunamended period is 0.0594 days⁻¹. The reaction rate constant for theamended period is 0.062 days⁻¹, or a 4.4 percent improvement. Thisimprovement occurred even though there was a 4.5° F. drop in temperatureduring the amended period. FIG. 19 gives the same data for Digester 2.As noted, k values for amended and unamended periods, found by fittingthe least squares line through the origin, were 0.0531 and 0.0487 day⁻¹,respectively. The improvement was 9.03 percent. The improvement ofDigester 2 was greater than Digester 1, however, the temperature ofDigester 2 was more stable.

                                      TABLE 32                                    __________________________________________________________________________    Chelating Agent Field Test, Weekly Average Performance                                                     Volatile Solids Fed                                                                         Volatile Solids Destroyed                       Vol. Solids                                                                           Feed Rate       (Kg/          (Kg/  Average                     Temp (°F.)                                                                   Conc. (g/L)                                                                           (gallons)                                                                             (Kg)    1000 gal)                                                                           (Kg)    1000                                                                                %al)                 Date   D1 D2 D1  D2  D1  D2  D1  D2  D1 D2 D1  D2  D1 D2 D1 D2                __________________________________________________________________________    BASELINE DATA                                                                  7-16 to 7-22                                                                        97.8                                                                             94.7                                                                             11.420                                                                            11.140                                                                            157300                                                                            183800                                                                            10029.4                                                                           11737.6                                                                           63.8                                                                             63.9                                                                             3234.8                                                                            3958.8                                                                            20.6                                                                             21.5                                                                             32.25                                                                            33.73              7-23 to 7-29                                                                        98.4                                                                             95.0                                                                             9.429                                                                             10.425                                                                            152400                                                                            200300                                                                            10967.0                                                                           14228.8                                                                           72.0                                                                             71.0                                                                             5646.4                                                                            6375.6                                                                            37.1                                                                             31.8                                                                             51.48                                                                            44.81              8-30 to 8-05                                                                        98.0                                                                             95.4                                                                             9.503                                                                             11.087                                                                            150300                                                                            174600                                                                            10440.8                                                                           12200.6                                                                           69.5                                                                             69.9                                                                             4351.6                                                                            3975.1                                                                            29.0                                                                             22.8                                                                             41.68                                                                            32.58              8-07 to 8-13                                                                        99.0                                                                             95.3                                                                             10.387                                                                            10.310                                                                            170600                                                                            187200                                                                            10726.7                                                                           11967.8                                                                           62.9                                                                             63.9                                                                             4250.6                                                                            3400.4                                                                            24.9                                                                             18.2                                                                             39.63                                                                            28.40               8-14 to 8-20                                                                       98.0                                                                             93.4                                                                             9.966                                                                             10.989                                                                            279300                                                                            258800                                                                            18758.1                                                                           16956.8                                                                           67.2                                                                             65.5                                                                             8292.6                                                                            6373.9                                                                            29.7                                                                             24.6                                                                             44.21                                                                            37.59              8-21 to 8-26                                                                        98.2                                                                             91.0                                                                             8.377                                                                             10.873                                                                            248100                                                                            216900                                                                            14736.5                                                                           12476.9                                                                           59.4                                                                             57.5                                                                             6935.6                                                                            3552.6                                                                            28.0                                                                             16.4                                                                             47.06                                                                            28.47             AMENDED DATA                                                                  NO AMENDMENT                                                                  11-13 to 11-15                                                                       90.3                                                                             87.0                                                                             8.489                                                                             10.439                                                                            92066                                                                             96030                                                                             8603.6                                                                            8998.1                                                                            93.5                                                                             93.7                                                                             5649.7                                                                            5200.1                                                                            61.4                                                                             54.2                                                                             65.67                                                                            57.79             D2 AMENDED                                                                    11-17 to 11-23                                                                       90.0                                                                             85.6                                                                             8.378                                                                             9.655                                                                             91602                                                                             137926                                                                            10829.1                                                                           16028.0                                                                           118.2                                                                            116.2                                                                            7909.9                                                                            10995.8                                                                           86.4                                                                             79.7                                                                             73.04                                                                            68.60             11-24 to 11-30                                                                       90.0                                                                             83.4                                                                             8.884                                                                             10.405                                                                            144293                                                                            147580                                                                            19203.0                                                                           19581.3                                                                           133.1                                                                            132.7                                                                            14186.0                                                                           13760.5                                                                           98.3                                                                             93.2                                                                             73.88                                                                            70.27             12-01 to 12-07                                                                       93.6                                                                             84.4                                                                             8.018                                                                             9.414                                                                             75674                                                                             103841                                                                            9804.5                                                                            13385.0                                                                           129.6                                                                            128.9                                                                            7502.6                                                                            9665.9                                                                            99.1                                                                             88.4                                                                             76.52                                                                            72.21             12-08 to 12-14                                                                       94.0                                                                             84.2                                                                             8.330                                                                             9.483                                                                             111587                                                                            166518                                                                            12507.6                                                                           18013.0                                                                           113.0                                                                            108.2                                                                            9088.4                                                                            12055.7                                                                           81.4                                                                             72.4                                                                             72.09                                                                            66.93             12-15 to 12-21                                                                       93.4                                                                             81.4                                                                             9.296                                                                             9.126                                                                             92835                                                                             80977                                                                             11898.5                                                                           10572.0                                                                           128.2                                                                            130.6                                                                            8990.5                                                                            7743.8                                                                            96.8                                                                             95.6                                                                             75.56                                                                            70.41             12-22 to 12-28                                                                       93.8                                                                             80.9                                                                             8.340                                                                             8.331                                                                             64462                                                                             39539                                                                             11726.4                                                                           7108.8                                                                            181.9                                                                            179.8                                                                            9690.7                                                                            5862.7                                                                            150.3                                                                            148.3                                                                            82.64                                                                            82.47             12-29 to 1-03                                                                        92.6                                                                             81.3                                                                             8.048                                                                             8.396                                                                             62080                                                                             48324                                                                             10368.9                                                                           8130.5                                                                            163.9                                                                            168.3                                                                            8297.9                                                                            6592.7                                                                            133.7                                                                            136.4                                                                            80.03                                                                            81.07             NO AMENDMENT                                                                   1-13 to 1-17                                                                        88.8                                                                             81.8                                                                             8.215                                                                             9.684                                                                             120169                                                                            117187                                                                            16303.2                                                                           15903.2                                                                           135.7                                                                            135.7                                                                            12562.7                                                                           11316.6                                                                           104.5                                                                            96.6                                                                             77.06                                                                            71.16             D1 AMENDED                                                                     1-18 to 1-24                                                                        86.4                                                                             81.6                                                                             8.863                                                                             10.191                                                                            116001                                                                            208289                                                                            14551.0                                                                           26171.4                                                                           114.9                                                                            111.2                                                                            10654.8                                                                           18137.2                                                                           91.9                                                                             87.1                                                                             73.22                                                                            69.30              1-25 to 1-31                                                                        88.6                                                                             83.0                                                                             9.429                                                                             10.887                                                                            124592                                                                            182441                                                                            17936.7                                                                           25618.5                                                                           144.0                                                                            140.4                                                                            13355.9                                                                           18244.1                                                                           107.2                                                                            99.3                                                                             74.46                                                                            71.21              2-1 to 2-7                                                                          88.0                                                                             82.0                                                                             11.751                                                                            11.572                                                                            116016                                                                            192859                                                                            10259.8                                                                           17057.0                                                                           88.4                                                                             88.4                                                                             4918.0                                                                            8607.5                                                                            42.4                                                                             44.6                                                                             47.93                                                                            50.46             28 to 2-14                                                                           87.7                                                                             82.4                                                                             8.841                                                                             11.820                                                                            122621                                                                            158765                                                                            16184.9                                                                           20881.2                                                                           131.9                                                                            131.5                                                                            12115.9                                                                           13778.1                                                                           98.8                                                                             86.8                                                                             74.91                                                                            65.98              2-15 to 2-21                                                                        89.6                                                                             83.1                                                                             7.579                                                                             12.047                                                                            118687                                                                            183939                                                                            11096.3                                                                           16810.2                                                                           93.5                                                                             91.4                                                                             7675.6                                                                            8431.0                                                                            64.7                                                                             45.8                                                                             69.17                                                                            50.15              2-22 to 2-28                                                                        89.6                                                                             85.5                                                                             8.709                                                                             12.038                                                                            88398                                                                             182143                                                                            10073.6                                                                           20755.6                                                                           114.0                                                                            144.0                                                                            7161.6                                                                            12460.1                                                                           81.0                                                                             68.4                                                                             71.09                                                                            60.03              2-29 to 3-6                                                                         86.0                                                                             86.7                                                                             8.506                                                                             11.778                                                                            82927                                                                             114950                                                                            9069.2                                                                            12558.5                                                                           109.4                                                                            109.3                                                                            6394.0                                                                            7443.3                                                                            77.1                                                                             64.8                                                                             70.50                                                                            59.27              3-7 to 3-13                                                                         89.8                                                                             87.3                                                                             8.146                                                                             11.461                                                                            135246                                                                            155413                                                                            13860.8                                                                           15460.8                                                                           102.5                                                                            99.3                                                                             9741.0                                                                            8694.7                                                                            71.6                                                                             55.9                                                                             70.28                                                                            56.33             __________________________________________________________________________

                                      TABLE 33                                    __________________________________________________________________________    Weekly Average Retention Time and Conversion of the Chelating Agent Field     Test                                                                                                     Inverse of                                                Average                                                                             Retention     Retention                                                 Temp (°F.)                                                                   Time, days                                                                          (CI-CO)/CO                                                                            Time, days-1                                                                          (CI-CO)/CI                                        D1 D2 D1 D2 D1  D2  D1  D2  D1  D2                                     __________________________________________________________________________    D2 AMENDED                                                                    11-17 to 11-23                                                                       90.0                                                                             85.6                                                                             52.2                                                                             38.9                                                                             2.727                                                                             2.180                                                                             0.0192                                                                            0.0257                                                                            0.7304                                                                            0.6860                                 11-24 to 11-30                                                                       90.0                                                                             83.4                                                                             33.1                                                                             36.4                                                                             2.958                                                                             2.369                                                                             0.0302                                                                            0.2075                                                                            0.7388                                                                            0.7027                                 12-01 to 12-07                                                                       93.6                                                                             84.4                                                                             63.2                                                                             51.7                                                                             3.270                                                                             2.618                                                                             0.0158                                                                            0.0193                                                                            0.7652                                                                            0.7221                                 12-08 to 12-14                                                                       94.0                                                                             84.2                                                                             42.9                                                                             32.2                                                                             2.584                                                                             2.015                                                                             0.0233                                                                            0.0311                                                                            0.7209                                                                            0.6693                                 12-15 to 12-21                                                                       93.4                                                                             81.4                                                                             51.5                                                                             66.3                                                                             3.083                                                                             2.781                                                                             0.0194                                                                            0.0151                                                                            0.7556                                                                            0.7041                                 12-22 to 12-28                                                                       93.8                                                                             80.9                                                                             74.2                                                                             135.8                                                                            4.762                                                                             4.702                                                                             0.0135                                                                            0.0074                                                                            0.8264                                                                            0.8247                                 12-29 to 1-03                                                                        92.6                                                                             81.3                                                                             66.0                                                                             95.3                                                                             4.381                                                                             4.296                                                                             0.0152                                                                            0.0105                                                                            0.8003                                                                            0.8107                                 D1 AMENDED                                                                     1-18 to 1-24                                                                        86.4                                                                             81.6                                                                             41.2                                                                             25.8                                                                             2.421                                                                             1.883                                                                             0.0243                                                                            0.0388                                                                            0.7322                                                                            0.6930                                  1-25 to 1-31                                                                        88.6                                                                             83.0                                                                             38.4                                                                             29.4                                                                             3.035                                                                             2.407                                                                             0.0260                                                                            0.0340                                                                            0.7446                                                                            0.7121                                  2-1 to 2-7                                                                          88.0                                                                             82.0                                                                             41.2                                                                             27.8                                                                             0.988                                                                             1.018                                                                             0.0243                                                                            0.0360                                                                            0.4793                                                                            0.5046                                  2-8 to 2-14                                                                         87.7                                                                             82.4                                                                             39.0                                                                             33.8                                                                             2.942                                                                             1.939                                                                             0.0256                                                                            0.0296                                                                            0.7491                                                                            0.6598                                  2-15 to 2-21                                                                        89.6                                                                             83.1                                                                             40.3                                                                             29.2                                                                             2.259                                                                             1.004                                                                             0.0248                                                                            0.0342                                                                            0.6917                                                                            0.5015                                  2-22 to 2-28                                                                        89.6                                                                             85.5                                                                             54.1                                                                             29.5                                                                             2.458                                                                             1.502                                                                             0.0185                                                                            0.0339                                                                            0.7109                                                                            0.6003                                  2-29 to 3-06                                                                        86.0                                                                             86.7                                                                             57.7                                                                             46.7                                                                             2.398                                                                             1.452                                                                             0.0173                                                                            0.0214                                                                            0.7050                                                                            0.5927                                  3-07 to 3-13                                                                        89.8                                                                             87.3                                                                             35.4                                                                             34.6                                                                             2.324                                                                             1.289                                                                             0.0282                                                                            0.0289                                                                            0.7028                                                                            0.5633                                 __________________________________________________________________________

Another means of studying the effect of chelator amendment is to plotvolatile solids conversion vs reciprocal retention time. The plots forDigesters 1 and 2 are shown in FIGS. 20 and 21, respectively. Linearrelationships are obtained for both digesters. The same slopes areobtained for the amended and unamended periods. However, higherconversions are obtained with chelation addition. For Digester 1, theimprovement is about 5 percent. For Digester 2, an improvement of 10.4percent over the unamended period was obtained. These results are veryclose to the improvements in the reaction rate contant (4.7% forDigester 1 and 9% for Digester 2).

It is concluded that the improvement of the 10 uM CA amendment is about10 percent. The lower 5% improvement in Digester 1 is probably theresult of a temperature drop of 4.5° F. These results compare reasonablywell with the laboratory studies, which showed an 18 percent improvementwith 10 uM CA. In view of the considerable variability and frequentupsets during the field tests, these improvements in rates areconsidered to be quite good.

EXAMPLE 12 Methanogen Production

The experiments were conducted by pressurizing the gas phase of 150 mlreactors to 20 psig with either N₂ or H₂ (80%) and CO₂ (20%), followedby inoculation with 50 ml of medium and 5 ml of Methanosarcina barkeri.Varying quantities of methanol, as the feed source, were added initiallyto the liquid phase. The reactors were thoroughly agitated in a 37° C.shaker incubator at 100 rpm. The composition and preparationinstructions of the medium used are given in Table 34.

                  TABLE 34                                                        ______________________________________                                        Minimal Medium for Methanogen Growth                                          Basic Solutions                                                                            Component       Concentration                                    ______________________________________                                        A. Basic Solutions                                                            I. Minerals A                                                                              K.sub.2 HPO.sub.4                                                                             6.0     g/L                                      II. Minerals KH.sub.2 PO.sub.4                                                                             6.0     g/L                                                   (NH.sub.4).sub.2 SO.sub.4                                                                     12.0    g/L                                                   NaCl            12.0    g/L                                                   MgSO.sub.4      2.6     g/L                                                   MgCl.sub.2      4.0     g/L                                      III. Trace Minerals                                                                        CoCl.sub.2      0.1     g/L                                                   Na.sub.2 MoO.sub.4                                                                            0.24    g/L                                                   NiCl.sub.2      0.24    g/L                                      IV. Vitamins biotin          2       mg/L                                                  folic acid      2       mg/L                                                  pyridoxine HCl  10      mg/L                                                  thiamine HCl    5       mg/L                                                  riboflavin      5       mg/L                                                  nicotinic acid  5       mg/L                                                  Ca-pantothenate 5       mg/L                                                  vitamin B-12    0.1     mg/L                                                  P-Amino benzoic acid                                                                          5       mg/L                                                  thioctic acid   5       mg/L                                     B. Preparation for One Liter of Methanogen Medium                             50 ml Basic Solution I, Minerals A                                            50 ml Basic Solution II, Minerals B                                           10 ml Basic Solution III, Trace Minerals                                      10 ml Basic Solution IV, Vitamins                                             2.7 g NH.sub.4 Cl                                                             2.5 g Na-Acetate                                                              5.0 g Na-bicarbonate                                                          0.01 g FeSO.sub.4                                                             1 ml of 1% resazurin                                                          Add distilled water to 1 L.                                                   Adjust pH to 6.9 with HCl solution                                            Boil and dispense under N.sub.2                                               Exchange head-space with 80% H.sub.2 and 20% CO.sub.2                         Autoclave                                                                     Reduce with 2.5% cysteine/sulfide                                             ______________________________________                                    

The resultant cell concentrations from these experiments are listed inTables 35 and 36. The reactors without H₂ and CO₂ did not grow to highconcentrations. Methanol, up to a concentration of 1.2 percent, is notan inhibitor. The cell concentration reaches a maximum of 15.25 g/Lafter 4 days. The results of these batch experiments show that H₂ andCO₂ are required for rapid growth and that the methanol concentrationshould not exceed 1.2 percent.

                  TABLE 35                                                        ______________________________________                                        Methanogen Batch Growth on Methanol                                           Under N.sub.2 or H.sub.2 and CO.sub.2                                         MeOH Concentration                                                                          Cell concentrations (g/l) after 4 days                          [percent]     N.sub.2      H.sub.2 and CO.sub.2                               ______________________________________                                        0             3.25         4.88                                               0.2           4.25         5.50                                               0.4           3.25         8.25                                               0.6           2.34         9.12                                               1.0           2.50         15.25                                              ______________________________________                                    

                  TABLE 36                                                        ______________________________________                                        Methanogen Batch Growth on Methanol                                           Under H.sub.2 and CO.sub.2                                                    MeOH Concentration                                                            (percent)      Day 1      Day 3   Day 5                                       ______________________________________                                        0              5.5        7.5     8.4                                         1.2            8.3        11.5    12.0                                        1.6            5.5        8.8     10.5                                        2.0            5.3        2.8     2.0                                         3.0            3.3        2.5     --                                          4.0            3.0        2.3     --                                          ______________________________________                                    

Fed-batch experiments were conducted in an attempt to increase the cellconcentration. Since both methanol and H₂ and CO₂ can be used as carbonand energy sources, the fed-batch experiments had to accommodate theaddition of both substrates. These experiments were conducted in 150 mlsealed reactors with 50 ml of medium and an inoculum of 5 mlMethanosarcina barkeri. Methanol, in concentrations of 0.6 to 1.0percent, was added at varying intervals, with H₂ and CO₂ replenishedevery one to three days. The results of adding H₂ and CO₂ every one,two, or three days are given in Tables 37, 38 and 39, respectively. Insome of these studies, methanogen media or concentrated media were alsoadded.

                  TABLE 37                                                        ______________________________________                                        Methanogen Growth Studies with H.sub.2                                        and CO.sub.2 Re-pressurized Every Day                                         Methanol Conc. Media                                                                              4       7    10    13   16                                Addition Addition   days    days days  days days                              ______________________________________                                        none     0          6.8     10.9 11.2  14.8 18.5                              (0.2%/day                                                                              0          12.5    14.8 15.0  17.0 12.3)                             0.6%/day 0          17.8    25.5 25.8  30.0 17.0                              1.0%/2 day                                                                             0          17.0    24.3 24.8  18.0 12.0                              0.6%/day 1 ml/day   15.5    23.5 24.3  26.3 25.8                              1.0%/2 day                                                                             1 ml/day   20.0    23.5 25.0  16.3 --                                1.0%/2 day                                                                             1 ml/2 day 17.0    23.0 21.5  20.0 9.3                               ______________________________________                                    

                  TABLE 38                                                        ______________________________________                                        Methanogen Growth Studies with H.sub.2 and                                    CO.sub.2 Re-pressurized Every Two Days                                        Methanol   Cell Concentration, (g/l)                                          Addition   3 days  6 days  9 days 12 days                                                                              15 days                              ______________________________________                                        none        6.3     9.3     9.5   11.0    8.8                                 1.0% once only                                                                           17.3    13.5    14.8   10.0                                        (0.2%/day  14.8(4) 18.8(7) 18.8(10)                                                                             16.3(13)                                                                             14.0)                                0.6%/day   13.0    29.8    33.5   37.5   37.0                                 1.0%/2 day 13.5    24.0    35.0   36.5   38.5                                 1.0%/3 day 12.8    24.5    24.0   27.8   26.0                                 ______________________________________                                    

                  TABLE 39                                                        ______________________________________                                        Methanogen Growth Studies with H.sub.2 and                                    CO.sub.2 Repressurized Every Three Days.                                      Methanol                                                                              Media                                                                 Addition                                                                              Addition    3 days  6 days 9 days                                                                              12 days                              ______________________________________                                        0.6%/day                                                                              0           4.8     7.3    5.0   5.8                                  1.0%/2 day                                                                            0           4.0     10.3   11.0  9.0                                  0.6%/day                                                                              5 ml/3 day  3.0     5.9    4.8   5.3                                  1.0%/2 day                                                                            5 ml/3 day  3.4     8.3    6.5   6.3                                  ______________________________________                                    

Further studies examined the effect of adding 1.0% methanol every twodays with gas addition every two days, adding 0.4% every day withre-pressurization every day, adding no methanol every day withre-pressurization every day, and adding 0.3 ml of 10% yeast extract atday 9 for each of these trials. The results are given in Table 40.

                  TABLE 40                                                        ______________________________________                                        Methanogen Growth Studies with H.sub.2 --CO.sub.2 Gas Cylinder                Methanol H.sub.2 --CO.sub.2                                                                       3       6    9     12   15                                Addition Addition   days    days days  days days                              ______________________________________                                        none     every day  8.68    12.00                                                                              16.67 22.08                                                                              25.83                             0.4%/day every day  24.00   24.75                                                                              27.68 38.34                                                                              28.33                             1.0%/2 day                                                                             every 2 days                                                                             19.25   35.00                                                                              38.71 40.41                                                                              43.75                             ______________________________________                                    

EXAMPLE 13 Methanogen Preservation

Methanosarcina barkeri was preserved by either freeze-drying orultra-freezing. The cells were prepared for preservation in sealedvessels with a H₂ --CO₂ environment to insure anaerobic conditions.Cryoprotective agents were added as follows: for freeze-drying, equalvolumes of 24 percent sucrose or 20 percent skim milk; forultra-freezing, 5 volume percent dimethyl sulfoxide (DMSO) or 10 volumepercent glycerol.

After preservation, the freeze dried cells were recovered in the samesealed vessel by injecting the original volume of medium. Ultra-freezersamples were rapidly thawed under moderate agitation in a 40° C. waterbath. The viability of the cultures was then measured by transferringone ml of culture into a 25 ml tube with 9 ml of medium. Successiveidentical transfers were made with identical dilution until no growth ofcells was detected. The tubes were provided 20 psig of H₂ --CO₂ forgrowth. The growth was measured by the volume of methane produced,determined by gas chromatography.

The preservation periods investigated included one week, and one, two,and three months. The results of the viability tests are listed in Table41. These results show that both the freeze-dried cells with sucrose andthe ultra-frozen cells have the potential to reach the viability ofunpreserved cells after three months storage. There are someinconsistencies in the viability reached at different preservationperiods for the same method. These problems could be caused by the shorttime for growth allowed in the one week and the one month tests. Inthese experiments, the investigation was stopped after six days, or thesame time that the unpreserved samples reached the maximum viability.The gas measurements were not conducted every day in these tests.However, for the two and three month experiments, the viability wastested every day for two weeks.

                  TABLE 41                                                        ______________________________________                                        The Viability of Methanogen Preservation                                              Viability                                                                       zero     one    one     two   three                                 Method    day      week   month   months                                                                              months                                ______________________________________                                        Un-preserved                                                                            10.sup.6                                                            Freeze-Drying                                                                 no protection      10.sup.2                                                                             --      10.sup.3                                    milk               10.sup.6                                                                             10.sup.6                                                                              10.sup.6                                                                            10.sup.4                              sucrose            10.sup.4                                                                             10.sup.5                                                                              10.sup.6                                                                            10.sup.6                              Ultra-Freezing                                                                no protection      10.sup.4                                                                             10.sup.5                                                                              10.sup.6                                                                            10.sup.6                              DMSO               10.sup.6                                                                             10.sup.6                                                                              10.sup.6                                                                            10.sup.6                              glycerol           10.sup.5                                                                             10.sup.6                                                                              10.sup.4                                                                            10.sup.6                              ______________________________________                                    

Viability is expressed as the maximum amount of culture dilution atwhich cell growth could be detected. For example, 10⁶ means that theculture was diluted by a factor of one million and cells were stillpresent.

The time required to reach a certain viability may provide a bettermeans for selection of the best preservation method. These results aregiven in Table 42. As shown, good results are obtained when cells arepreserved by ultra-freezing with DMSO, which gives the fastest responseand is very close to the result of the unpreserved samples. Protectionwith glycerol was almost as good; however, the performance in the twomonth test gave only 10⁴ viability. Interestingly, cells withoutcryprotection recover from ultra-freezing, but only about half asquickly. Preservation by freeze-drying was not as effective as byultra-freezing.

                                      TABLE 42                                    __________________________________________________________________________    Days Required to Reach Full Viability of Methanosarcina barkeri                       2 Month Preservation                                                                            3 Month Preservation                                Viability                                                                             10.sup.1                                                                         10.sup.2                                                                         10.sup.3                                                                         10.sup.4                                                                         10.sup.5                                                                         10.sup.6                                                                         10.sup.1                                                                         10.sup.2                                                                         10.sup.3                                                                         10.sup.4                                                                         10.sup.5                                                                         10.sup.6                             __________________________________________________________________________    Freeze-Drying                                                                 No protection                                                                         4  8  10 -- -- --                                                     Skim Milk                                                                             1  1  3  5  7  10 1  2  4  6  -- --                                   Sucrose 1  2  3  5  6  10 1  2  5  6  10 10                                   Ultra-Freezing                                                                No protection                                                                         1  1  2  6  6  7  1  1  2  7  9  10                                   DMSO    1  1  1  3  4  5  1  1  2  3  4  5                                    Glycerol                                                                              1  1  4  8  -- -- 1  1  2  5  5  6                                    Un-preserved                                                                          1  1  1  (3)                                                                              (4)                                                                              5  1  1  1  (3)                                                                              (4)                                                                              5                                    __________________________________________________________________________     note: () estimated.                                                      

I claim:
 1. A method for improved performance of anaerobic solidsdigesters comprising the addition of at least one chelating agent to asolid substrate for solubilizing solid nutrients to enhance bacterialgrowth.
 2. The method of claim 1, further comprising the addition of atleast one nutrient.
 3. The method according to claim 2, wherein thechelating agent is selected from the group consisting of 1:2diaminocyclonexane-N, N-tetra acetic acid (CDTA), ethylenedimaine tetraacetic acid (EDTA), citric acid (CA), and nitrilo trieacetic acid (NTA).4. The method according to claim 2, wherein the chelating agentcomprises nitrilo triscetic acid (NTA).
 5. The method according to claim2, wherein the nutrient is selected from the group consisting of iron,sulfide, selenium, and nickel.
 6. The method according to claim 2,wherein the nutrient comprises FESO₄.
 7. The method according to claim2, wherein the nutrients are selected from the group consisting ofFESO₄, FeCl₂, SeO₂, and NiCl₂.
 8. The method according to claim 2,wherein the dosage of said chelating agent is approximately 10 uM. 9.The method according to claim 2, wherein the dosage of said chelatingagent ranges about 1 uM to about 100 uM.
 10. The method according toclaim 2, wherein the concentration of nutrients is approximately 10 uM.11. The method according to claim 2, wherein the concentration ofnutrients ranges from about 1 uM to about 5000 uM.
 12. The methodaccording to claim 1, wherein the chelating agent is selected from thegroup consisting of 1:2 diaminocyclonexane-N, N-tetra acetic acid(CDTA), ethylenediamine tetra acetic acid (EDTA), citric acid (CA), andnitrilo triacetic acid (NTA).
 13. The method according to claim 1,wherein the chelating agent comprises nitrilo triacetic acid (NTA). 14.The method according to claim 1, wherein the dosage of said chelatingagent is approximately 10 uM.
 15. The method according to claim 1,wherein the dosage of said chelating agent ranges about 1 uM to about100 uM.
 16. A method for improved performance of anaerobic solidsdigesters comprising the addition of a chelating agent comprising 1:2diamino cyclohexane-N, N-tetra acetic acid (CDTA) to a solid substratefor solubilizing solid nutrients to enhance bacterial growth incombination with the addition of a nutrient comprising FESO₄.